Patent Application
20200386748
Provided herein are systems and methods for the detection of an analyte or analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and/or quantifying a target analyte using a bioluminescent complex comprising substrates, peptides, and/or polypeptides capable of generating a bioluminescent signal that correlates to the presence, absence, or amount of the target analyte.
Biological processes rely on covalent and non-covalent interactions between molecules, macromolecules, and molecular complexes. In order to understand such processes, and to develop techniques and compounds to manipulate them for research and clinical and other practical applications, it is necessary to have tools available to detect and monitor these interactions and/or components involved in such interactions. The study of these interactions, particularly under physiological conditions (e.g., at normal expression levels for monitoring protein interactions), requires high sensitivity.
Creation of better assays for use in the field and in clinical settings is an ongoing area of urgent need. Speed, sensitivity, selectivity, robustness, simplicity, quantitative versus qualitative capabilities, and cost are all critical factors affecting the relevance of a diagnostic bioassays, and thus their utility to and adoption by the relevant community. Rapid diagnostic tests are not only relevant to clinical settings, but also can be applied to environmental, industrial, and direct to consumer contexts.
Provided herein are compositions and formulations comprising a luminogenic substrate and a target analyte binding agent comprising a target analyte binding element and one of a polypeptide component of a bioluminescent complex, or a peptide component of a bioluminescent complex.
In accordance with these embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 5; at least 60% sequence identity with SEQ ID NO: 9; or at least 60% sequence identity with SEQ ID NO: 12.
In some embodiments, the peptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 10; at least 60% sequence identity with SEQ ID NO: 11; at least 60% sequence identity with SEQ ID NO: 13; or at least 60% sequence identity with SEQ ID NO: 14.
In some embodiments, the composition comprises a complementary peptide or polypeptide component of the bioluminescent complex, wherein the target analyte binding agent and the complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of a target analyte.
In some embodiments, the composition that comprises the luminogenic substrate and the target analyte binding agent are combined in a dried formulation, and the complementary peptide or polypeptide component of the bioluminescent complex comprises a liquid formulation, wherein the liquid formulation is added to the dried formulation and forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the composition comprising the luminogenic substrate, the target analyte binding agent, and the complementary peptide or polypeptide component of the bioluminescent complex are combined in a dried formulation, wherein the dried formulation forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the complementary peptide or polypeptide component comprises a second target analyte binding element that forms the bioluminescent analyte detection complex in the presence of the target analyte.
In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 6, and wherein the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity with SEQ ID NO: 10.
In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 6, and wherein the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity with SEQ ID NO: 14.
Embodiments of the present disclosure also include a composition comprising a dried formulation comprising (a) a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 9, and (b) a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 10.
In some embodiments, the dried formulation further comprises a luminogenic substrate.
In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.
Embodiments of the present disclosure also include a composition comprising a dried formulation comprising (a) a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and (b) a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 14.
In some embodiments, the dried formulation further comprises a luminogenic substrate.
In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.
Embodiments of the present disclosure also include a composition comprising a dried formulation comprising (a) a first target analyte binding agent comprising a first target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, (b) a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15, and (c) a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 12.
In some embodiments, the dried formulation further comprises a luminogenic substrate.
In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.
Embodiments of the present disclosure also include a composition comprising (a) a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 9, and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11.
Embodiments of the present disclosure also include a composition comprising (a) a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11, and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 9.
Embodiments of the present disclosure also include a composition comprising (a) a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 14.
In some embodiments, the dried formulation further comprises a luminogenic substrate.
In some embodiments, the liquid formulation further comprises a luminogenic substrate.
In some embodiments, the liquid formulation further comprises a sample comprising a target analyte, and wherein a bioluminescent analyte detection complex forms upon combining the dried formulation and the liquid formulation in the presence of the target analyte.
In some embodiments, the composition further comprises a second complementary peptide or polypeptide component of the bioluminescent complex, wherein the target analyte binding agent, the first complementary peptide or polypeptide component of the bioluminescent complex, and the second complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of a target analyte.
In some embodiments, the composition comprising the target analyte binding agent comprises a dried formulation, and wherein the first complementary peptide or polypeptide component and the second complementary peptide or polypeptide of the bioluminescent complex comprise a liquid formulation; wherein the liquid formulation is added to the dried formulation and forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the composition comprising the target analyte binding agent, and either the first or the second complementary peptide or polypeptide component are combined in a dried formulation, and wherein the first or the second complementary peptide or polypeptide component that is not present in the dried formulation comprises a liquid formulation; wherein the liquid formulation is added to the dried formulation and forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the target analyte binding agent, the first complementary peptide or polypeptide component, and the second complementary peptide or polypeptide component are combined in a dried formulation that forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the dried formulation further comprises a luminogenic substrate.
In some embodiments, the liquid formulation further comprises a luminogenic substrate.
In some embodiments, the liquid formulation further comprises a sample comprising a target analyte, and wherein a bioluminescent analyte detection complex forms upon combining the dried formulation and the liquid formulation in the presence of the target analyte.
In some embodiments, either the first or the second complementary peptide or polypeptide component comprises a second target analyte binding element that forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 6, and wherein either the first or the second complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity with either SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include a composition comprising (a) a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 6, and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and a second complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include (a) a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 6, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and (b) a liquid formulation comprising a second complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include (a) a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 6, and complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include (a) a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15, and (b) a liquid formulation comprising a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 6.
Embodiments of the present disclosure also include (a) a dried formulation comprising a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 6, and (b) a liquid formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15.
Embodiments of the present disclosure also include a composition comprising a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15, and a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 6.
In some embodiments, the dried formulation further comprises a luminogenic substrate.
In some embodiments, the liquid formulation further comprises a luminogenic substrate.
In some embodiments, the liquid formulation further comprises a sample comprising a target analyte, and wherein a bioluminescent analyte detection complex forms upon combining the dried formulation and the liquid formulation in the presence of the target analyte.
In some embodiments, a bioluminescent signal produced in the presence of the luminogenic substrate is substantially increased when the target analyte binding agent contacts one or more of the complementary peptide or polypeptide components of the bioluminescent complex, as compared to a bioluminescent signal produced by the target analyte binding agent and the luminogenic substrate alone.
In some embodiments, the target analyte is a target antibody.
In some embodiments, the target analyte binding agent comprises an element that binds non-specifically to antibodies.
In some embodiments, the target analyte binding agent comprises an element that binds specifically to an antibody.
In some embodiments, the target antibody is an antibody against a pathogen, toxin, or therapeutic biologic.
In some embodiments, a target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, protein A, an Ig binding domain of protein A, protein G, an Ig binding domain of protein G, protein A/G, an Ig binding domain of protein A/G, protein L, a Ig binding domain of protein L, protein M, an Ig binding domain of protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.
In some embodiments, the luminogenic substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW, 1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives.
In some embodiments, the composition further comprises a polymer.
In some embodiments, the polymer is a naturally-occurring biopolymer. In some embodiments, the naturally-occurring biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran, and a combination of any thereof. In some embodiments, the naturally-occurring biopolymer is pullulan.
In some embodiments, the polymer is a cyclic saccharide polymer or a derivative thereof. In some embodiments, the polymer is hydroxypropyl β-cyclodextrin.
In some embodiments, the polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly(propylene oxide) block and at least one poly(ethylene oxide) block. In some embodiments, the synthetic polymer is poloxamer 188.
In some embodiments, the composition further comprises a substance to reduce autoluminescence.
In some embodiments, the substance to reduce autoluminescence is ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like.
In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a chelating agent, a protein, or any combination thereof. In some embodiments, the is surfactant selected from polysorbate 20, polysorbate 40, and polysorbate 80.
In some embodiments, the composition is used in conjunction with an analyte detection platform to detect an analyte in a sample.
In some embodiments, sample is selected from blood, serum, plasma, urine, stool, cerebral spinal fluid, interstitial fluid, saliva, a tissue sample, a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample.
Embodiments of the present disclosure also include a method of detecting an analyte in a sample comprising combining any of the compositions described above with a sample comprising a target analyte.
In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from an analyte detection complex.
In some embodiments, the method further comprises quantifying a bioluminescent signal generated from the analyte detection complex.
In some embodiments, the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte.
In some embodiments, one or more of the components of the composition exhibits enhanced stability within the composition compared to the component in solution alone.
Embodiments of the present disclosure also include systems and methods for the detection of an analyte or analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and/or quantifying a target analyte using a bioluminescent complex comprising substrates, peptides, and/or polypeptides capable of generating a bioluminescent signal that correlates to the presence, absence, or amount of the target analyte.
Embodiments of the present disclosure include a lateral flow detection system. In accordance with these embodiments, the system includes an analytical membrane that includes a detection region and a control region. In some embodiments, the detection region includes a first target analyte binding agent immobilized to the detection region, a conjugate pad comprising a second target analyte binding agent, and a sample pad. In some embodiments, the first target analyte binding agent and the second target analyte binding agent form a bioluminescent analyte detection complex in the at least one detection region when a target analyte is detected in a sample.
In some embodiments, the first target analyte binding agent includes a target analyte binding element and is non-luminescent. In some embodiments, the second target analyte binding agent includes a target analyte binding element and a bioluminescent polypeptide. In some embodiments, the bioluminescent polypeptide has at least 60% sequence identity with SEQ ID NO: 5.
In some embodiments, the first target analyte binding agent includes a target analyte binding element and a polypeptide component of a bioluminescent complex, and the second target analyte binding agent includes a target analyte binding element and a peptide component of a bioluminescent complex. In some embodiments, a bioluminescent signal produced in the presence of a luminogenic substrate is substantially increased when the first target analyte binding agent contacts the second target analyte binding agent, as compared to a bioluminescent signal produced by the first target analyte binding agent and the luminogenic substrate alone.
In some embodiments, the first target analyte binding agent includes a target analyte binding element and a peptide component of a bioluminescent complex, and the second target analyte binding agent includes a target analyte binding element and a polypeptide component of a bioluminescent complex. In some embodiments, a bioluminescent signal produced in the presence of a luminogenic substrate is substantially increased when the first target analyte binding agent contacts the second target analyte binding agent, as compared to a bioluminescent signal produced by the first target analyte binding agent and the luminogenic substrate alone.
In some embodiments, the polypeptide component of a bioluminescent complex has at least 60% sequence identity with SEQ ID NO: 6. In some embodiments, the polypeptide component of a bioluminescent complex has at least 60% sequence identity with SEQ ID NO: 10. In some embodiments, the polypeptide component of a bioluminescent complex has at least 60% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide component of a bioluminescent complex has at least 60% sequence identity with SEQ ID NO: 14.
In some embodiments, the first target analyte binding agent includes a target analyte binding element and a first peptide component of a tripartite bioluminescent complex, and the second target analyte binding agent includes a target analyte binding element and a second peptide component of the tripartite bioluminescent complex. In some embodiments, a bioluminescent signal produced in the presence of a luminogenic substrate is substantially increased when the first target analyte binding agent contacts the second target analyte binding agent and a polypeptide component of the tripartite bioluminescent complex as compared to a bioluminescent signal produced by (i) the first target analyte binding agent, the second target analyte binding agent, and/or the polypeptide component and (ii) the luminogenic substrate alone.
In some embodiments, the first peptide component of a tripartite bioluminescent complex has at least 60% sequence identity with SEQ ID NO: 11. In some embodiments, the second first peptide component of a tripartite bioluminescent complex has at least 60% sequence identity with SEQ ID NO: 13. In some embodiments, the polypeptide component of a tripartite bioluminescent complex has at least 60% sequence identity with SEQ ID NO: 12.
In some embodiments, the target analyte is a target antibody. In some embodiments, the first target analyte binding element includes an agent that binds non-specifically to antibodies. In some embodiments, the second target analyte binding element comprises an agent that binds specifically to the target antibody. In some embodiments, the target antibody is an antibody against a pathogen, toxin, or therapeutic biologic.
In some embodiments, a target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, protein A, an Ig binding domain of protein A, protein G, an Ig binding domain of protein G, protein A/G, an Ig binding domain of protein A/G, protein L, a Ig binding domain of protein L, protein M, an Ig binding domain of protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.
In some embodiments, the system further includes a luminogenic substrate. In some embodiments, the luminogenic substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the luminogenic substrate is applied to the system as part of a composition that includes the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the luminogenic substrate is applied to the system as part of a composition that includes the luminogenic substrate and a substance to reduce autoluminescence such as ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like.
In some embodiments, the composition is applied to at least one of the sample pad, the conjugation pad, the detection region, and the control region.
In some embodiments, the analytical membrane includes a plurality of detection regions with each detection region comprising a distinct target analyte binding agent having distinct target analyte binding elements.
In some embodiments, the system further includes a device for detecting or quantifying bioluminescent signals from the analyte detection complex.
Embodiments of the present disclosure also include a conjugate pad comprising at least one target analyte binding agent. In accordance with these embodiments, the at least one target analyte binding agent includes a target analyte binding element and one of: a bioluminescent polypeptide comprising at least 60% sequence identity with SEQ ID NO: 5; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 9; a peptide comprising at least 60% sequence identity with SEQ ID NO: 10; a peptide comprising at least 60% sequence identity with SEQ ID NO: 11; a peptide comprising at least 60% sequence identity with SEQ ID NO: 13; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 12; a peptide comprising at least 60% sequence identity with SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the target analyte binding agent includes a target analyte binding element and one of: a bioluminescent polypeptide of SEQ ID NO: 5; a polypeptide of SEQ ID NO: 9; a peptide of SEQ ID NO: 10; a peptide of SEQ ID NO: 11; a peptide of SEQ ID NO: 13; a polypeptide of SEQ ID NO: 12; a peptide of SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the conjugate pad further includes a luminogenic substrate. In some embodiments, the luminogenic substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the luminogenic substrate contained on or within the conjugate pad as part of a composition that includes the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the luminogenic substrate is applied to the system as part of a composition that includes the luminogenic substrate and a substance to reduce autoluminescence such as ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like.
Embodiments of the present disclosure also include an analytical membrane that includes a detection region and a control region. In accordance with these embodiments, the detection region includes at least one target analyte binding agent immobilized to the detection region.
In some embodiments, the at least one target analyte binding agent includes a target analyte binding element and one of: a bioluminescent polypeptide comprising at least 60% sequence identity with SEQ ID NO: 5; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 9; a peptide comprising at least 60% sequence identity with SEQ ID NO: 10; a peptide comprising at least 60% sequence identity with SEQ ID NO: 11; a peptide comprising at least 60% sequence identity with SEQ ID NO: 13; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 12; a peptide comprising at least 60% sequence identity with SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the target analyte binding agent includes a target analyte binding element and one of: a bioluminescent polypeptide of SEQ ID NO: 5; a polypeptide of SEQ ID NO: 9; a peptide of SEQ ID NO: 10; a peptide of SEQ ID NO: 11; a peptide of SEQ ID NO: 13; a polypeptide of SEQ ID NO: 12; a peptide of SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the analytical membrane further includes a plurality of detection regions with each detection region comprising a distinct target analyte binding agent having distinct target analyte binding elements. In some embodiments, the analytical membrane further includes a luminogenic substrate. In some embodiments, the luminogenic substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives.
In some embodiments, the luminogenic substrate is reversibly conjugated to the conjugate pad as part of a composition including the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the luminogenic substrate is part of a composition that includes the luminogenic substrate and a substance that reduces autoluminescence such as ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like.
Embodiments of the present disclosure also include a solid phase detection platform comprising a detection region. In accordance with these embodiments, the detection region includes at least one target analyte binding agent conjugated to the detection region. In some embodiments, the at least one target analyte binding agent includes a target analyte binding element and one of: a bioluminescent polypeptide comprising at least 60% sequence identity with SEQ ID NO: 5; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 9; a peptide comprising at least 60% sequence identity with SEQ ID NO: 10; a peptide comprising at least 60% sequence identity with SEQ ID NO: 11; a peptide comprising at least 60% sequence identity with SEQ ID NO: 13; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 12; a peptide comprising at least 60% sequence identity with SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the target analyte binding agent includes a target analyte binding element and one of: a bioluminescent polypeptide of SEQ ID NO: 5; a polypeptide of SEQ ID NO: 9; a peptide of SEQ ID NO: 10; a peptide of SEQ ID NO: 11; a peptide of SEQ ID NO: 13; a polypeptide of SEQ ID NO: 12; a peptide of SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the detection platform includes: a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 6 conjugated to the detection region; and a second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity with SEQ ID NO: 10 applied to the detection region.
In some embodiments, the detection platform includes: a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 10 conjugated to the detection region; and a second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity with SEQ ID NO: 6 applied to the detection region.
In some embodiments, the detection platform includes: a first target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity with SEQ ID NO: 11 conjugated to the detection region; a second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity with SEQ ID NO: 13 applied to the detection region; and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 12 applied to the detection region.
In some embodiments, the detection platform includes: a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 6 conjugated to the detection region; and a second target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with ID NO: 14 applied to the detection region.
In some embodiments, the detection platform includes: a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 14 conjugated to the detection region; and a second target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 6 applied to the detection region.
In some embodiments, the detection platform includes: a first target analyte binding agent comprising a target analyte binding element and a bioluminescent polypeptide at least 60% sequence identity with SEQ ID NO: 5 conjugated to the detection region; and a second target analyte binding agent comprising a target analyte binding element and a fluorophore capable of being activated by energy transfer from the bioluminescent polypeptide applied to the detection region.
In some embodiments, the detection platform includes: a first target analyte binding agent comprising a target analyte binding element and a bioluminescent polypeptide at least 60% sequence identity with SEQ ID NO: 5 applied to the detection region; and a second target analyte binding agent comprising a target analyte binding element and a fluorophore capable of being activated by energy transfer from the bioluminescent polypeptide conjugated to the detection region.
In some embodiments, the detection platform further includes a plurality of detection regions with each detection region comprising a distinct target analyte binding agent having distinct target analyte binding elements. In some embodiments, the detection platform further includes a control region. In some embodiments, the detection platform further includes a luminogenic substrate. In some embodiments, the luminogenic substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the luminogenic substrate is reversibly conjugated to the conjugate pad as part of a composition comprising the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the luminogenic substrate is part of a composition comprising the luminogenic substrate and a substance that reduces autoluminescence such as ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like.
Embodiments of the present disclosure also include a solution phase detection platform that includes at least one detection receptacle and a lyophilized tablet (lyocake). In accordance with these embodiments, the lyocake comprises a target analyte binding agent comprising a target analyte binding element and one of: a bioluminescent polypeptide comprising at least 60% sequence identity with SEQ ID NO: 5; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 9; a peptide comprising at least 60% sequence identity with SEQ ID NO: 10; a peptide comprising at least 60% sequence identity with SEQ ID NO: 11; a peptide comprising at least 60% sequence identity with SEQ ID NO: 13; a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 12; a peptide comprising at least 60% sequence identity with SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the target analyte binding agent comprises a target analyte binding element and one of: a bioluminescent polypeptide of SEQ ID NO: 5; a polypeptide of SEQ ID NO: 9; a peptide of SEQ ID NO: 10; a peptide of SEQ ID NO: 11; a peptide of SEQ ID NO: 13; a polypeptide of SEQ ID NO: 12; a peptide of SEQ ID NO: 14; or a fluorophore capable of being activated by energy transfer from an Oplophorus luciferase.
In some embodiments, the lyocake comprises: a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 6; and a second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity with SEQ ID NO: 10.
In some embodiments, the lyocake comprises: a first target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity with SEQ ID NO: 11; a second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity with SEQ ID NO: 13; and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 12.
In some embodiments, the lyocake comprises: a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with SEQ ID NO: 6; and a second target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity with ID NO: 14.
In some embodiments, the lyocake comprises: a first target analyte binding agent comprising a target analyte binding element and a bioluminescent polypeptide at least 60% sequence identity with SEQ ID NO: 5; and a second target analyte binding agent comprising a target analyte binding element and a fluorophore capable of being activated by energy transfer from the bioluminescent polypeptide.
In some embodiments, the detection platform comprises a 96-well microtiter plate comprising a plurality of detection receptacles, and at least two distinct target analyte binding agents comprising distinct target analyte binding elements.
In some embodiments, the lyocake comprises a luminogenic substrate. In some embodiments, the luminogenic substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives.
In some embodiments, the lyocake comprises a luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof.
In some embodiments, the lyocake comprises a luminogenic substrate and a substance to reduce autoluminescence such as ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like.
In some embodiments, the detection platform further comprises at least one sample. In some embodiments, the sample is selected from blood, serum, plasma, urine, stool, cerebral spinal fluid, interstitial fluid, saliva, a tissue sample, a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample.
Embodiments of the present disclosure also include a method of detecting an analyte in a sample using the lateral flow assay systems described above. In accordance with these embodiments, the method includes applying a sample to the sample pad, facilitating flow of the sample from the sample pad to the conjugate pad, and then from the conjugate pad to the detection region and the control region on the analytical membrane. In some embodiments, the first target analyte binding agent, the second target analyte binding agent, and the target analyte form the analyte detection complex in the at least one detection region when the target analyte is detected in the sample.
In some embodiments, the sample is a sample from a subject selected from blood, serum, plasma, urine, stool, cerebral spinal fluid, interstitial fluid, tissue, and saliva. In some embodiments, the sample is selected from a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample. In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from the analyte detection complex.
In some embodiments, the method further comprises quantifying a bioluminescent signal generated from the analyte detection complex. In some embodiments, the method further comprises diagnosing a subject from which the sample was obtained as having or not having a disease based on the detection of the analyte.
Embodiments of the present disclosure also include a method of detecting an analyte in a sample using the solid phase detection platform described above. In accordance with these embodiments, the method includes exposing a sample to the detection region and control region. In some embodiments, the at least one target analyte binding agent and the at least one target analyte form an analyte detection complex in the at least one detection region when the target analyte is detected in the sample.
In some embodiments, the sample is a sample from a subject selected from blood, serum, plasma, urine, stool, cerebral spinal fluid, interstitial fluid, tissue, and saliva. In some embodiments, the sample is selected from a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample. In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from the analyte detection complex.
In some embodiments, the method further comprises quantifying a bioluminescent signal generated from the analyte detection complex. In some embodiments, the method further comprises diagnosing a subject from which the sample was obtained as having or not having a disease based on the detection of the analyte.
Embodiments of the present disclosure also include a method of producing a substrate for use in a bioluminescent assay. In accordance with these embodiments, the method includes applying a solution onto a substrate. In some embodiments, the solution contains at least one target analyte binding agent comprising a target analyte binding element and one of a polypeptide component of a bioluminescent complex or a peptide component of a bioluminescent complex. In some embodiments, the method includes drying the substrate containing the solution.
In some embodiments, the solution further includes a complementary peptide or polypeptide component of the bioluminescent complex. In some embodiments, the target analyte binding agent and the complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of a target analyte.
In some embodiments, the solution comprises a protein buffer and at least one excipient. In some embodiments, the solution comprises a luminogenic substrate.
In some embodiments, the substrate comprising the dried solution is W-903 paper, FTA paper, FTA Elute paper, FTA DMPK paper, Ahlstrom A-226 paper, M-TFN paper, FTA paper, FP705 paper, Bode DNA collection paper, nitrocellulose paper, nylon paper, cellulose paper, Dacron paper, cotton paper, and polyester papers, or combinations thereof. In some embodiments, the substrate is a mesh comprising plastic, nylon, metal, or combinations thereof.
In some embodiments, drying the substrate containing the solution comprises drying at a temperature from about 30° C. to 40° C. for a period of time between about 30 mins and 2 hours. In some embodiments, drying the substrate containing the solution comprises lyophilizing and/or freezing the substrate.
In some embodiments, the method further comprises drying the at least one target analyte binding agent and/or the complementary peptide or polypeptide component of the bioluminescent complex onto a first substrate, and drying the luminogenic substrate onto a second substrate.
In accordance with these embodiments, a bioluminescent signal is generated upon exposure of the substrate containing the solution to the target analyte, and in some embodiments, the bioluminescent signal is proportional to the concentration of the target analyte.
In some embodiments, the at least one target analyte binding agent and/or the complementary peptide or polypeptide component of the bioluminescent complex exhibit(s) enhanced stability when dried on the substrate.
Embodiments of the present disclosure include a composition comprising a luminogenic substrate, a target analyte binding agent comprising a target analyte binding element and a polypeptide component of a bioluminescent complex, and a complementary polypeptide component of the bioluminescent complex. In accordance with these embodiments, the target analyte binding agent and the complementary polypeptide component of the bioluminescent complex are capable of forming a bioluminescent analyte detection complex in the presence of a target analyte.
In some embodiments, the composition further comprises a second target analyte binding agent comprising a second target analyte binding element and a second polypeptide component of a bioluminescent complex.
In some embodiments, the first and second target analyte binding agents bind separate portions of the same target analyte.
In some embodiments, the first and second polypeptide components of the bioluminescent complex bind the complementary polypeptide component of the bioluminescent complex to form a bioluminescent analyte detection complex in the presence of the target analyte.
In some embodiments, the first and the second polypeptide components are linked to a modified dehalogenase capable of forming a covalent bond with a haloalkane substrate.
In some embodiments, the first and the second target analyte binding elements comprise a haloalkane substrate.
In some embodiments, the first or second polypeptide components of the first and second target analyte binding agents comprise: at least 60% sequence identity with SEQ ID NO: 10; at least 60% sequence identity with SEQ ID NO: 11; at least 60% sequence identity with SEQ ID NO: 13; or at least 60% sequence identity with SEQ ID NO: 15.
In some embodiments, the complementary polypeptide component comprises: at least 60% sequence identity with SEQ ID NO: 6; at least 60% sequence identity with SEQ ID NO: 9; or at least 60% sequence identity with SEQ ID NO: 12.
In some embodiments, the target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, protein A, an Ig binding domain of protein A, protein G, an Ig binding domain of protein G, protein A/G, an Ig binding domain of protein A/G, protein L, a Ig binding domain of protein L, protein M, an Ig binding domain of protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.
In some embodiments, the target analyte is an antibody, and wherein the target analyte binding element of the first target analyte binding agent comprises antigen recognized by the antibody, and wherein the target analyte binding element of the second target analyte binding agent comprises an Fc binding region.
In some embodiments, the first and/or second target analyte binding agents further comprise a fluorophore coupled to the first and/or second polypeptide components of the bioluminescent complex.
In some embodiments, one or more components of the composition is in the form of a lyophilized tablet (lyocake) capable of forming a bioluminescent complex when reconstituted in a solution to detect and/or quantify the target analyte.
In some embodiments, the composition comprises a solution-phase detection platform capable of detecting and/or quantifying the target analyte.
In some embodiments, the polypeptide components and the luminogenic substrate are in the form of a lyophilized tablet (lyocake) capable of forming a bioluminescent complex when reconstituted in a solution to detect and/or quantify the target analyte.
Embodiments of the present disclosure also includes a method of detecting an analyte in a sample comprising combining any of the compositions described above with a sample comprising a target analyte.
In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from an analyte detection complex.
In some embodiments, the method further comprises quantifying a bioluminescent signal generated from the analyte detection complex.
In some embodiments, the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
Embodiments of the present disclosure provide systems and methods for the detection of an analyte or analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and/or quantifying a target analyte using a bioluminescent complex comprising substrates, peptides, and/or polypeptides capable of generating a bioluminescent signal that correlates to the presence, absence, or amount of the target analyte.
Most rapid diagnostic bioassays are based on immunological principles. Some embodiments of the present disclosure combine immunoassay-based concepts with the advantages of bioluminescence, which include a large linear range and extremely low background, among other advantages. However, despite these advantages, point-of-care bioluminescence-based immunoassays are not yet commercially available. Some reasons for this may be that many currently available luciferases have low signal, which inherently limits their usefulness in immunoassays. Additionally, when a bioluminescent signal output is configured to be conditional (e.g., through complementation or bioluminescence resonance energy transfer (BRET)), the signal can be reduced even further. Many currently available luciferases also have a low tolerance or sensitivity to certain assay conditions, such as high temperatures, non-optimal buffer compositions, and complex sample matrices, thus requiring specialized chemistries to be compatible with point-of-care devices.
Embodiments of the present disclosure also address the need for “all-in-one” assay formats for analyte detection, which until the present application, have not been developed or described in the prior art. For example, Tenda, K. et al. (Angew. Chem. Int. Ed. 57, 15369—15373 (2018)) discloses paper devices where the substrate and bioluminescent components are dried onto separate sections of the paper, rather than being included together in a single-format system. Additionally, Yu, Q. et al. (Science 361, 1122-1126 (2018)) discloses that, although the bioluminescent components can be dried together, the substrate is separately mixed with the analyte-of-interest and subsequently added to the paper rather than drying the substrate and the bioluminescent components in a single format system. As described further herein, embodiments of the present disclosure provide methods, compositions, and systems that include all the necessary components of a bioluminescent detection complex (excluding the analyte-of-interest) in a single-format (e.g., “all-in-one”) system. This contrasts with currently available systems, which include at least one of the necessary bioluminescent components in a separate format/solution. Thus, embodiments of the present disclosure provide surprising and unexpected advantages over currently available bioluminescent analyte detection systems.
To address the need for bioluminescent-based point-of-care immunoassay platforms that are not necessarily limited to the use of typical immunoassay reagents, embodiments of the present disclosure include the use of the NanoLuc® bioluminescent platform, including compositions and methods for the assembly of a bioluminescent complex from two or more peptide and/or polypeptide components. In some embodiments, the peptide and/or polypeptide components are not fragments of a preexisting protein (e.g., are not complementary subsequences of a known polypeptide sequence), but confer bioluminescent activity via structural complementation (See, e.g., WO/2014/151736 (Intl. App. No. PCT/US2014/026354) and U.S. patent application Ser. No. 16/439,565 (PCT/US2019/036844), herein incorporated by reference in their entireties), as described further herein. In some embodiments, peptide and/or polypeptide components are non-luminescent in the absence of complementation and/or complementation enhances bioluminescence of a peptide or polypeptide component. In some embodiments, target analyte binding agents are labeled with the various components of the bioluminescent complexes described herein without comprising the ability of the binding agents to bind their target analytes. Components of the bioluminescent complexes of the present disclosure are configured to be compatible with currently available point-of-care devices and systems such as lateral flow devices, paper-based spot tests, dip stick tests, lab-on-a-chip, microfluidic devices, pre-filled 96-well microtiter plates, and the like.
For example, embodiments of the present disclosure incorporate NanoLuc®-based technologies (e.g., NanoBiT, NanoTrip, Nano-Glo (e.g., NANOGLO Live Cell Substrate or NANOGLO LCS (Promega Cat. Nos. N205 and N113)), NanoBRET, etc.) into target analyte detection assays that can be embedded in a solid phase assay or device, including plastics, matrices, and membranes of various composition, and/or used in other assay formats such as lyophilized cakes or tablets for solution phase assays, all of which function reliably even in complex sampling environments (e.g., blood components, food matrix, soil samples, stool, urine, water, and other human and animal biological samples). In some embodiments, NanoLuc®-based reporter systems are incorporated into lateral flow assay (LFA) technology, paper spot tests, and similar devices. LFAs are a commonly used point-of-care technology used to measure a variety of target analytes including, but not limited to, antibodies, bacterial and viral antigens, metabolites, proteins, and the like. As demonstrated in
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
“Bioluminescence” refers to production and emission of light by a chemical reaction catalyzed by, or enabled by, an enzyme, protein, protein complex, or other biomolecule (e.g., bioluminescent complex). In typical embodiments, a substrate for a bioluminescent entity (e.g., bioluminescent protein or bioluminescent complex) is converted into an unstable form by the bioluminescent entity; the substrate subsequently emits light.
“Complementary” refers to the characteristic of two or more structural elements (e.g., peptide, polypeptide, nucleic acid, small molecule, etc.) of being able to hybridize, dimerize, or otherwise form a complex with each other. For example, a “complementary peptide and polypeptide” are capable of coming together to form a complex. Complementary elements may require assistance to form a complex (e.g., from interaction elements), for example, to place the elements in the proper conformation for complementarity, to co-localize complementary elements, to lower interaction energy for complementation, etc.
“Complex” refers to an assemblage or aggregate of molecules (e.g., peptides, polypeptides, etc.) in direct and/or indirect contact with one another. In one aspect, “contact,” or more particularly, “direct contact” means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules. In such an aspect, a complex of molecules (e.g., a peptide and polypeptide) is formed under assay conditions such that the complex is thermodynamically favored (e.g., compared to a non-aggregated, or non-complexed, state of its component molecules). As used herein the term “complex,” unless described as otherwise, refers to the assemblage of two or more molecules (e.g., peptides, polypeptides or a combination thereof).
“Derivative” of an antibody as used herein may refer to an antibody having one or more modifications to its amino acid sequence when compared to a genuine or parent antibody and exhibit a modified domain structure. The derivative may still be able to adopt the typical domain configuration found in native antibodies, as well as an amino acid sequence, which is able to bind to targets (antigens) with specificity. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or fragments of antibodies. The derivative may also comprise at least one further compound, such as a protein domain linked by covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art. The additional domain present in the fusion protein comprising the antibody may preferably be linked by a flexible linker, advantageously a peptide linker, wherein said peptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of the further protein domain and the N-terminal end of the antibody or vice versa. The antibody may be linked to an effector molecule having a conformation suitable for biological activity or selective binding to a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug, for example.
“Fragment” refers to a peptide or polypeptide that results from dissection or “fragmentation” of a larger whole entity (e.g., protein, polypeptide, enzyme, etc.), or a peptide or polypeptide prepared to have the same sequence as such. Therefore, a fragment is a subsequence of the whole entity (e.g., protein, polypeptide, enzyme, etc.) from which it is made and/or designed. A peptide or polypeptide that is not a subsequence of a preexisting whole protein is not a fragment (e.g., not a fragment of a preexisting protein). A peptide or polypeptide that is “not a fragment of a preexisting bioluminescent protein” is an amino acid chain that is not a subsequence of a protein (e.g., natural or synthetic) that: (1) was in physical existence prior to design and/or synthesis of the peptide or polypeptide, and (2) exhibits substantial bioluminescent activity.
As used herein, the term “antibody fragment” refers to a portion of a full-length antibody, including at least a portion of the antigen binding region or a variable region. Antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody) produced by recombinant DNA techniques, or chemical polypeptide synthesis. For example, a “Fab” fragment comprises one light chain and the CH1 and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab′” fragment comprises one light chain and one heavy chain that comprises additional constant region, extending between the CH1 and CH2 domains. An interchain disulfide bond can be formed between two heavy chains of a Fab′ fragment to form a “F(ab′)2” molecule. An “Fv” fragment comprises the variable regions from both the heavy and light chains, but lacks the constant regions. A single-chain Fv (scFv) fragment comprises heavy and light chain variable regions connected by a flexible linker to form a single polypeptide chain with an antigen-binding region. Exemplary single chain antibodies are discussed in detail in WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203; herein incorporated by reference in their entireties. In certain instances, a single variable region (e.g., a heavy chain variable region or a light chain variable region) may have the ability to recognize and bind antigen. Other antibody fragments will be understood by skilled artisans.
“Isolated polynucleotide” as used herein may mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, by virtue of its origin, the isolated polynucleotide is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
“Non-luminescent” refers to an entity (e.g., peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting a detectable amount of light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be referred to as non-luminescent if it does not exhibit detectable luminescence in a given assay. As used herein, the term “non-luminescent” is synonymous with the term “substantially non-luminescent. For example, a non-luminescent polypeptide is substantially non-luminescent, exhibiting, for example, a 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×103-fold, 1×104-fold, 1×105-fold, 1×106-fold, 1×107-fold, etc.) reduction in luminescence compared to a complex of the polypeptide with its non-luminescent complement peptide. In some embodiments, an entity is “non-luminescent” if any light emission is sufficiently minimal so as not to create interfering background for a particular assay.
“Non-luminescent peptide” and “non-luminescent polypeptide” refer to peptides and polypeptides that exhibit substantially no luminescence (e.g., in the presence of a substrate), or an amount that is beneath the noise, or a 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×103-fold, 1×104-fold, 1×105-fold, 1×106-fold, 1×107-fold, etc.) when compared to a significant signal (e.g., luminescent complex) under standard conditions (e.g., physiological conditions, assay conditions, etc.) and with typical instrumentation (e.g., luminometer, etc.). In some embodiments, such non-luminescent peptides and polypeptides assemble, according to the criteria described herein, to form a bioluminescent complex. As used herein, a “non-luminescent element” is a non-luminescent peptide or non-luminescent polypeptide. The term “bioluminescent complex” refers to the assembled complex of two or more non-luminescent peptides and/or non-luminescent polypeptides. The bioluminescent complex catalyzes or enables the conversion of a substrate for the bioluminescent complex into an unstable form; the substrate subsequently emits light. When uncomplexed, two non-luminescent elements that form a bioluminescent complex may be referred to as a “non-luminescent pair.” If a bioluminescent complex is formed by three or more non-luminescent peptides and/or non-luminescent polypeptides, the uncomplexed constituents of the bioluminescent complex may be referred to as a “non-luminescent group.”
“Peptide” and “polypeptide” as used herein, and unless otherwise specified, refer to polymer compounds of two or more amino acids joined through the main chain by peptide amide bonds (—C(O)NH—). The term “peptide” typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), whereas the term “polypeptide” typically refers to longer amino acid polymers (e.g., chains having more than 25 amino acids).
“Preexisting protein” refers to an amino acid sequence that was in physical existence prior to a certain event or date. A “peptide that is not a fragment of a preexisting protein” is a short amino acid chain that is not a fragment or sub-sequence of a protein (e.g., synthetic or naturally-occurring) that was in physical existence prior to the design and/or synthesis of the peptide.
“Sample,” “test sample,” “specimen,” “sample from a subject,” and “patient sample” as used herein may be used interchangeable and may be a sample of blood, such as whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
“Sequence identity” refers to the degree two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families, e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.
“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal and a human. In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing forms of treatment. “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, llamas, camels, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits, guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
“Subsequence” refers to peptide or polypeptide that has 100% sequence identify with another, larger peptide or polypeptide. The subsequence is a perfect sequence match for a portion of the larger amino acid chain.
“Substantially” as used herein means that the recited characteristic, parameter, and/or value need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement error, measurement accuracy limitations and other factors known to skill in the art, may occur in amounts that do not preclude the effect the characteristic was intended to provide. A characteristic or feature that is substantially absent (e.g., substantially non-luminescent) may be one that is within the noise, beneath background, below the detection capabilities of the assay being used, or a small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%) of the significant characteristic (e.g., luminescent intensity of a bioluminescent protein or bioluminescent complex).
“Variant” is used herein to describe a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. “SNP” refers to a variant that is a single nucleotide polymorphism. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response. Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid (e.g., replacing an amino acid with a different amino acid of similar properties, such as hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
“Target analyte” or “analyte” as used herein refers to a substance in a sample that can be detected, quantified, measured, tested, and/or monitored, often as part of a method of evaluating a process or condition (e.g., diagnostic or prognostic assay). Target analytes can include, but are not limited to, a protein, a peptide, a polypeptide, an enzyme, a cofactor, a nucleotide, a polynucleotide, DNA, RNA, a small molecule compound, an antibody, and any variation, combination, and derivative thereof.
“Target analyte binding agent” as used herein refers to an agent capable of binding to a target analyte. In some embodiments, target analyte binding agents include agents that can bind multiple substances, such as a target analyte and a solid phase support. In some embodiments, target analyte binding agents include agents that bind both a target analyte (e.g., via a target analyte binding element) and a distinct peptide/polypeptide to form a target analyte detection complex (e.g., to generate a bioluminescent signal). In some embodiments, target analyte binding agents can include target analyte binding elements capable of binding a group or class of analytes (e.g., protein L binding to antibodies); and in other embodiments, target analyte binding agents can include target analyte binding elements capable of binding a specific analyte (e.g., an antigen binding a monoclonal antibody). A target analyte binding agent may be an antibody, antibody fragment, a receptor domain that binds a target ligand, proteins or protein domains that bind to immunoglobulins (e.g., protein A, protein G, protein A/G, protein L, protein M), a binding domain of a proteins that bind to immunoglobulins (e.g., protein A, protein G, protein A/G, protein L, protein M), oligonucleotide probe, peptide nucleic acid, DARPin, aptamer, affimer, a purified protein, or a protein domain (either the analyte itself or a protein that binds to the analyte), and analyte binding domain(s) of proteins etc. Table A provides a lists of exemplary binding moieties that could be used singly or in various combinations in methods, systems, and assays (e.g., immunoassays) herein.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
The present disclosure includes materials and methods related to bioluminescent polypeptides, bioluminescent complexes and components thereof, and bioluminescence resonance energy transfer (BRET).
In some embodiments, provided herein are solid phase and/or lateral flow assays, devices, and systems incorporating bioluminescent polypeptides and/or bioluminescent complexes (of non-luminescent peptide(s) and/or non-luminescent polypeptide components) based on (e.g., structurally, functionally, etc.) the luciferase of Oplophorus gracilirostris, the NanoLuc® luciferase (Promega Corporation; U.S. Pat. Nos. 8,557,970; 8,669,103; herein incorporated by reference in their entireties), the NanoBiT (U.S. Pat. No. 9,797,889; herein incorporated by reference in its entirety), or NanoTrip (U.S. patent application Ser. No. 16/439,565; and U.S. Prov. Appln. Ser. No. 62/941,255; both of which are herein incorporated by reference in their entireties). As described below, in some embodiments, the compositions, assays, devices, methods, and systems herein incorporate commercially available NanoLuc®-based technologies (e.g., NanoLuc® luciferase, NanoBRET, NanoBiT, NanoTrip, NanoGlo, etc.), but in other embodiments, various combinations, variations, or derivations from the commercially available NanoLuc®-based technologies are employed.
a. NanoLuc
PCT Appln. No. PCT/US2010/033449, U.S. Pat. No. 8,557,970, PCT Appln. No. PCT/2011/059018, and U.S. Pat. No. 8,669,103 (each of which is herein incorporated by reference in their entirety and for all purposes) describe compositions and methods comprising bioluminescent polypeptides. Such polypeptides find use in embodiments herein and can be used in conjunction with the compositions, assays, devices, systems, and methods described herein.
In some embodiments, compositions, assays, devices, systems, and methods provided herein comprise a bioluminescent polypeptide of SEQ ID NO: 5, or having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 5.
In some embodiments, any of the aforementioned bioluminescent polypeptides are linked (e.g., fused, chemically linked, etc.) to a binding element or other component of the assays and systems described herein.
In some embodiments, any of the aforementioned bioluminescent polypeptides, or fusions or conjugates thereof (e.g., with a binding element, etc.), are immobilized to a portion of a device described herein (e.g., a detection or control region of a lateral flow assay, a solid phase detection element, etc.).
b. NanoBiT
PCT Appln. No. PCT/US14/26354 and U.S. Pat. No. 9,797,889 (each of which is herein incorporated by reference in their entirety and for all purposes) describe compositions and methods for the assembly of bioluminescent complexes; such complexes, and the peptide and polypeptide components thereof, find use in embodiments herein and can be used in conjunction with the assays and methods described herein.
In some embodiments, provided herein are non-luminescent (NL) polypeptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 9, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 6.
In some embodiments, provided herein are non-luminescent (NL) peptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 10, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 8.
In some embodiments, provided herein are NL peptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 8.
In some embodiments, any of the aforementioned NL peptides or NL polypeptides are linked (e.g., fused, chemically linked, etc.) to a binding element or other component of the composition, assays, devices, methods, and systems described herein.
In some embodiments, any of the aforementioned NL peptides or NL polypeptides, or fusions or conjugates thereof (e.g., with a binding element, etc.), are immobilized to a portion of a device described herein (e.g., a detection or control region of a lateral flow assay, a solid phase detection element, etc.).
In some embodiments, provided herein is a lateral flow detection system comprising: an analytical membrane comprising a detection region and a control region, wherein the detection region comprises a first target analyte binding agent immobilized to the detection region; a conjugate pad comprising a second target analyte binding agent; and a sample pad; wherein the first target analyte binding agent comprises a first target analyte binding element and a first NanoBiT-based NL peptide or NL polypeptide component (as described above), and wherein the second target analyte binding agent comprises a second target analyte binding element and a complementary NanoBiT-based NL peptide or NL polypeptide component (as described above). In some embodiments, the first target analyte binding agent and the second target analyte binding agent form an analyte detection complex in the at least one detection region when a target analyte is detected in a sample. In some embodiments, a bioluminescent signal produced in the presence of a luminogenic substrate is substantially increased when the first target analyte binding agent contacts the second target analyte binding agent, as compared to a bioluminescent signal produced by the second target analyte binding agent or the first target analyte binding agent and the luminogenic substrate alone.
In some embodiments, provided herein is solid-phase detection system comprising: an solid phase substrate comprising a first target analyte binding agent and a second target analyte binding agent; wherein the first target analyte binding agent comprises a first target analyte binding element and a first NanoBiT-based NL peptide or NL polypeptide component (as described above), and wherein the second target analyte binding agent comprises a second target analyte binding element and a complementary NanoBiT-based NL peptide or NL polypeptide component (as described above). In some embodiments, the first target analyte binding agent and the second target analyte binding agent form an analyte detection complex in the solid-phase substrate when a target analyte is detected in a sample. In some embodiments, a bioluminescent signal produced in the presence of a luminogenic substrate is substantially increased when the first target analyte binding agent contacts the second target analyte binding agent, as compared to a bioluminescent signal produced by the second target analyte binding agent or the first target analyte binding agent and the luminogenic substrate alone.
c. NanoTrip
U.S. patent application Ser. No. 16/439,565 (PCT/US2019/036844) and U.S. Prov. Appln. Ser. No. 62/941,255 (both of which are herein incorporated by reference in their entireties and for all purposes) describes compositions, systems, and methods for the assembly of bioluminescent complexes. Such complexes, and the peptides and polypeptide components thereof, find use in embodiments herein and can be used in conjunction with the assays and methods described herein.
In some embodiments, provided herein are non-luminescent (NL) polypeptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 12, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 9.
In some embodiments, provided herein are non-luminescent (NL) peptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 8.
In some embodiments, provided herein are NL peptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 13, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 7.
In some embodiments, provided herein are NL peptides having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity with SEQ ID NO: 14, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8.
In some embodiments, any of the aforementioned NanoTrip-based NL peptide or NL polypeptides are linked (e.g., fused, chemically linked, etc.) to a binding element or other component of the compositions, methods, devices, assays, and systems described herein.
In some embodiments, any of the aforementioned NanoTrip-based NL peptide or NL polypeptides, or fusions or conjugates thereof (e.g., with a binding element, etc.), are immobilized to a portion of a device described herein (e.g., a detection or control region of a lateral flow assay, a solid phase detection element, etc.).
In some embodiments, provided herein is a lateral flow detection system comprising: an analytical membrane comprising a detection region and a control region, wherein the detection region comprises a first target analyte binding agent immobilized to the detection region; a conjugate pad comprising a second target analyte binding agent; and a sample pad; wherein the first target analyte binding agent comprises a first target analyte binding element and a first NanoTrip-based NL peptide (as described above), and wherein the second target analyte binding agent comprises a second target analyte binding element and a complementary NanoTrip-based NL peptide (as described above). In some embodiments, the first target analyte binding agent and the second target analyte binding agent form an analyte detection complex in the at least one detection region in the presence of a NanoTrip-based NL polypeptide component (as described above) when a target analyte is detected in a sample. In some embodiments, a bioluminescent signal produced in the presence of a luminogenic substrate is substantially increased when the first target analyte binding agent contacts the second target analyte binding agent in the presence of a NanoTrip-based NL polypeptide component, as compared to a bioluminescent signal produced by the second target analyte binding agent or the first target analyte binding agent and the luminogenic substrate alone.
In some embodiments, provided herein is a solid-phase detection system comprising: a solid phase (e.g., paper substrate, etc.) comprising a first target analyte binding agent and a second target analyte binding agent, wherein the first target analyte binding agent comprises a first target analyte binding element and a first NanoTrip-based NL peptide (as described above), and wherein the second target analyte binding agent comprises a second target analyte binding element and a complementary, second NL NanoTrip-based peptide (as described above). In some embodiments, the first target analyte binding agent and the second target analyte binding agent form an analyte detection complex in the presence of a NanoTrip-based NL polypeptide (as described above) when a target analyte is detected in a sample. In some embodiments, a bioluminescent signal produced in the presence of a luminogenic substrate is substantially increased when the first target analyte binding agent contacts the second target analyte binding agent and a NanoTrip-based NL polypeptide, as compared to a bioluminescent signal produced by the second target analyte binding agent or the first target analyte binding agent and the luminogenic substrate alone.
d. NanoBRET
As disclosed in PCT Appln. No. PCT/US13/74765 and U.S. patent application Ser. No. 15/263,416 (herein incorporated by reference in their entireties and for all purposes) describe bioluminescence resonance energy transfer (BRET) compositions, systems, and methods (e.g., incorporating NanoLuc®-based technologies); such compositions, systems and methods, and the bioluminescent polypeptide and fluorophore-conjugated components thereof, find use in embodiments herein and can be used in conjunction with the compositions, systems, devices, assays, and methods described herein.
In some embodiments, any of the NanoLuc®-based, NanoBiT-based, and/or NanoTrip-based (described in sections a-c, above) peptides, polypeptide, complexes, fusions, and conjugates may find use in BRET-based applications with the compositions, assays, methods, devices, and systems described herein. For example, in certain embodiments, a first target analyte binding agent comprises a first target analyte binding element and a NanoLuc®-based, NanoBiT-based, and/or NanoTrip-based polypeptide, peptide, or complex, and a second target analyte binding agent comprises a second target analyte binding element and a fluorophore (e.g., fluorescent protein, small molecule fluorophore, etc.), wherein the emission spectrum of the NanoLuc®-based, NanoBiT-based, and/or NanoTrip-based polypeptide, peptide, or complex overlaps the excitation spectrum of the fluorophore. In some embodiments, the NanoLuc®-based, NanoBiT-based, and/or NanoTrip-based polypeptide, peptide, or complex can be prepared in lyophilized form, which can include, or not include, the luminogenic substrate (e.g., furimazine).
In some embodiments, a target analyte binding agent comprises a target analyte binding element and a fluorophore capable of being activated by energy transfer from a bioluminescent polypeptide.
As used herein, the term “energy acceptor” refers to any small molecule (e.g., chromophore), macromolecule (e.g., autofluorescent protein, phycobiliproteins, nanoparticle, surface, etc.), or molecular complex that produces a readily detectable signal in response to energy absorption (e.g., resonance energy transfer). In certain embodiments, an energy acceptor is a fluorophore or other detectable chromophore. Suitable fluorophores include, but are not limited to: xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, Texas red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphin, phtalocyanine, bilirubin, etc.), CF dye (Biotium), BODIPY (Invitrogen), ALEXA FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes)(Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, etc. In some embodiments, a fluorophore is a rhodamine analog (e.g., carboxy rhodamine analog), such as those described in U.S. patent application Ser. No. 13/682,589, herein incorporated by reference in its entirety.
e. HALOTAG
Some embodiments herein comprise a capture protein capable of forming a covalent bond with a capture ligand. The capture protein may be linked to a first element (e.g., a peptide component of a bioluminescent complex) and the capture ligand to a second element (e.g., a target analyte binding element (e.g., an antibody or antigen binding protein)) and the formation of a covalent bond links the first and second elements to each other. In some embodiments, linking the first and second elements creates a target analyte binding agent. In some embodiments, two or more target analyte binding agents so formed can bind to a complementary polypeptide component (e.g., LgTrip) and form a bioluminescent complex in the presence of an analyte (e.g., a target antigen recognized by the target analyte binding element) (See e.g.,
One such modified dehalogenase is the commercially-available HALOTAG protein (SEQ ID NO: 720). In some embodiments, a capture protein comprises a polypeptide with at least 70% sequence identity (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 98% identity, 99% identity) with SEQ ID NO.: 720. Some embodiment comprise a fusion protein of the capture protein (e.g., HALOTAG) and another peptide/polypeptide element (e.g., a binding moiety, a peptide/polypeptide component of a bioluminescent complex, etc.). In some embodiments, a capture ligand is a haloalkane comprising a halogen (e.g., Cl, Br, F, I, etc.) covalently attached to the end of an alkyl chain (e.g., (CH2)4-24). In some embodiments, the other end of the alkyl chain is attached to a linker or to another element (e.g., a peptide, analyte, etc.). A linker may comprise an alkyl chain or substituted alkyl chain (e.g., C═O, NH, S, O, carbamate, ethylene etc.) such as those disclosed in U.S. patent application Ser. No. 14/207,959, herein incorporated by reference.
Embodiments of the present disclosure include compositions and formulations comprising one or more of the peptide and/or polypeptide components of the bioluminescent complexes provided herein. In accordance with these embodiments, compositions and formulations of the present disclosure can include a luminogenic substrate and/or various other components. The compositions and methods provided herein can be used to formulate shelf-stable liquid formulations (e.g., used in a solution phase assay format) and shelf-stable dried formulations (e.g., used in a solid phase assay format) capable of producing a luminescent signal in the presence of an analyte-of-interest, even after storage for prolonged time periods. As described further below, the compositions and formulations of the present disclosure can include one or more components of NanoLuc, NanoBiT, NanoTrip, and NanoBRET as well as the various luminogenic substrates described herein (e.g., furimazine).
In contrast to many currently available fluorescent and colorimetric assays, the compositions and formulations of the present disclosure provide means for conducting bioassays in which one or more of the peptide and/or polypeptide components of the bioluminescent complexes exist in a stable, dried formulation that is capable of being reconstituted in a solution containing, for example, a complementary peptide/polypeptide and/or a luminogenic substrate, such that the bioluminescent complex is formed in the presence of the analyte-of-interest. In some embodiments, the compositions and formulations of the present disclosure provide the means for conducting robust solid phase bioassays in which the bioluminescent signal produced is quantitative and proportional to the concentration of the analyte-of-interest.
In some embodiments, the compositions and formulations of the present disclosure include a luminogenic substrate and a target analyte binding agent that includes a target analyte binding element and a polypeptide component of a bioluminescent complex or a peptide component of a bioluminescent complex. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 6, at least 60% sequence identity with SEQ ID NO: 9, or at least 60% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 70% sequence identity with SEQ ID NO: 6, at least 70% sequence identity with SEQ ID NO: 9, or at least 70% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 80% sequence identity with SEQ ID NO: 6, at least 80% sequence identity with SEQ ID NO: 9, or at least 80% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 85% sequence identity with SEQ ID NO: 6, at least 85% sequence identity with SEQ ID NO: 9, or at least 85% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 90% sequence identity with SEQ ID NO: 6, at least 90% sequence identity with SEQ ID NO: 9, or at least 90% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 95% sequence identity with SEQ ID NO: 6, at least 95% sequence identity with SEQ ID NO: 9, or at least 95% sequence identity with SEQ ID NO: 12.
In other embodiments, the peptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 10, at least 60% sequence identity with SEQ ID NO: 11, at least 60% sequence identity with SEQ ID NO: 13, or at least 60% sequence identity with SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 70% sequence identity with SEQ ID NO: 10, at least 70% sequence identity with SEQ ID NO: 11, at least 70% sequence identity with SEQ ID NO: 13, or at least 70% sequence identity with SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 80% sequence identity with SEQ ID NO: 10, at least 80% sequence identity with SEQ ID NO: 11, at least 80% sequence identity with SEQ ID NO: 13, or at least 80% sequence identity with SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 85% sequence identity with SEQ ID NO: 10, at least 85% sequence identity with SEQ ID NO: 11, at least 85% sequence identity with SEQ ID NO: 13, or at least 85% sequence identity with SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 90% sequence identity with SEQ ID NO: 10, at least 90% sequence identity with SEQ ID NO: 11, at least 90% sequence identity with SEQ ID NO: 13, or at least 90% sequence identity with SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 95% sequence identity with SEQ ID NO: 10, at least 95% sequence identity with SEQ ID NO: 11, at least 95% sequence identity with SEQ ID NO: 13, or at least 95% sequence identity with SEQ ID NO: 14.
In some embodiments, the composition or formulation comprises a complementary peptide or polypeptide component of the bioluminescent complex. In accordance with these embodiments, the target analyte binding agent and the complementary peptide or polypeptide component of the bioluminescent complex can form a bioluminescent analyte detection complex in the presence of a target analyte. In some embodiments, the composition that comprises the luminogenic substrate and the target analyte binding agent can be combined in a dried formulation, and the complementary peptide or polypeptide component of the bioluminescent complex can be formulated as a liquid formulation. In some embodiments, the liquid formulation is added to the dried formulation and forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration. In other embodiments, the composition or formulation comprising the luminogenic substrate, the target analyte binding agent, and the complementary peptide or polypeptide component of the bioluminescent complex are combined in a dried formulation, wherein the dried formulation forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the complementary peptide or polypeptide component comprises a second target analyte binding element that forms the bioluminescent analyte detection complex in the presence of the target analyte. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 6, and wherein the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity with SEQ ID NO: 10. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 6, and wherein the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity with SEQ ID NO: 14.
Embodiments of the present disclosure also include a composition or formulation comprising a dried formulation that includes a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 9, and a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 10. In some embodiments, the dried formulation further comprises a luminogenic substrate. In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.
Embodiments of the present disclosure also include a composition comprising a dried formulation that includes a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 14. In some embodiments, the dried formulation further comprises a luminogenic substrate. In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.
Embodiments of the present disclosure also include a composition comprising a dried formulation that includes a first target analyte binding agent comprising a first target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15, and a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 12. In some embodiments, the dried formulation further comprises a luminogenic substrate. In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.
Embodiments of the present disclosure also include a composition that includes a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 9, and a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11.
Embodiments of the present disclosure also include a composition that includes a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 11, and a liquid formulation that contains a second target analyte binding agent comprising a target analyte binding element and a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 9.
Embodiments of the present disclosure also include a composition that includes a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 14. In some embodiments, the dried formulation further comprises a luminogenic substrate. In some embodiments, the liquid formulation further comprises a luminogenic substrate. In some embodiments, the liquid formulation further includes a sample comprising a target analyte. In accordance with these embodiments, a bioluminescent analyte detection complex forms upon combining the dried formulation and the liquid formulation in the presence of the target analyte.
In some embodiments, the composition further comprises a second complementary peptide or polypeptide component of the bioluminescent complex. In accordance with these embodiments, the target analyte binding agent, the first complementary peptide or polypeptide component of the bioluminescent complex, and the second complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of a target analyte.
In some embodiments, the composition comprising the target analyte binding agent are produced as a dried formulation. In some embodiments, the first complementary peptide or polypeptide component and the second complementary peptide or polypeptide of the bioluminescent complex are produced as a liquid formulation. In accordance with these embodiments, the liquid formulation can be added to the dried formulation, which facilitates the formation of the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the composition comprising the target analyte binding agent, and either the first or the second complementary peptide or polypeptide component are combined in a dried formulation, and the first or the second complementary peptide or polypeptide component that is not present in the dried formulation are produced as a liquid formulation. The liquid formulation can be added to the dried formulation, which facilitates the formation of the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the target analyte binding agent, the first complementary peptide or polypeptide component, and the second complementary peptide or polypeptide component are combined in a dried formulation that forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration. In some embodiments, the dried formulation further comprises a luminogenic substrate. In some embodiments, the liquid formulation further comprises a luminogenic substrate. In some embodiments, the liquid formulation further comprises a sample comprising a target analyte, wherein a bioluminescent analyte detection complex forms upon combining the dried formulation and the liquid formulation in the presence of the target analyte.
In some embodiments, either the first or the second complementary peptide or polypeptide component comprises a second target analyte binding element that forms the bioluminescent analyte detection complex in the presence of the target analyte upon rehydration.
In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity with SEQ ID NO: 12, and wherein either the first or the second complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity with either SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include a composition that includes a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and further including a second complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and further including a liquid formulation comprising a second complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15, and a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 13 or SEQ ID NO: 15.
Embodiments of the present disclosure also include a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15, and further including a liquid formulation comprising a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 12.
Embodiments of the present disclosure also include a dried formulation comprising a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 12, and a liquid formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15.
Embodiments of the present disclosure also include a composition comprising a dried formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity with SEQ ID NO: 13, a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity with SEQ ID NO: 15, and a complementary polypeptide component having at least 60% sequence identity with SEQ ID NO: 12. In some embodiments, the dried formulation further comprises a luminogenic substrate. In some embodiments, the liquid formulation further comprises a luminogenic substrate. In some embodiments, the liquid formulation further comprises a sample comprising a target analyte, and wherein a bioluminescent analyte detection complex forms upon combining the dried formulation and the liquid formulation in the presence of the target analyte.
In some embodiments, a bioluminescent signal produced in the presence of the luminogenic substrate is substantially increased when the target analyte binding agent contacts one or more of the complementary peptide or polypeptide components of the bioluminescent complex, as compared to a bioluminescent signal produced by the target analyte binding agent and the luminogenic substrate alone.
In some embodiments, the target analyte is a target antibody. In some embodiments, the target analyte binding agent comprises an element that binds non-specifically to antibodies. In some embodiments, the target analyte binding agent comprises an element that binds specifically to an antibody. In some embodiments, the target antibody is an antibody against a pathogen, toxin, or therapeutic biologic.
In some embodiments, a target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, protein A, an Ig binding domain of protein A, protein G, an Ig binding domain of protein G, protein A/G, an Ig binding domain of protein A/G, protein L, a Ig binding domain of protein L, protein M, an Ig binding domain of protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.
In some embodiments, the luminogenic substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the coelenterazine analogs or derivatives are pro-luminogenic substrates such as those disclosed in U.S. Pat. No. 9,487,520, herein incorporated by reference. In some embodiments, the coelenterazine analogs or derivatives are Enduazine (Promega Corporation) and Vivazine (Promega Corporation).
In some embodiments, the composition further comprises a polymer. In some embodiments, the polymer is a naturally-occurring biopolymer. In some embodiments, the naturally-occurring biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran, and a combination of any thereof. In some embodiments, the naturally-occurring biopolymer is pullulan. In some embodiments, the polymer is a cyclic saccharide polymer or a derivative thereof. In some embodiments, the polymer is hydroxypropyl β-cyclodextrin.
In some embodiments, the polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly(propylene oxide) block and at least one poly(ethylene oxide) block. In some embodiments, the synthetic polymer is poloxamer 188.
In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a chelating agent, a protein, or any combination thereof. In some embodiments, the is surfactant selected from polysorbate 20, polysorbate 40, and polysorbate 80.
In some embodiments, the composition further comprises a substance that reduces autoluminescence. In some embodiments, the substance is ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like. In some embodiments, the substance is a thionucleoside disclosed in U.S. Pat. No. 9,676,997, herein incorporated by reference. In some embodiments, the substance is thiourea, which use for reducing autoluminescence is disclosed in U.S. Pat. Nos. 7,118,878; 7,078,181; and 7,108,996, herein incorporated by reference.
In some embodiments, the composition is used in conjunction with an analyte detection platform to detect an analyte in a sample. In some embodiments, sample is selected from blood, serum, plasma, urine, stool, cerebral spinal fluid, interstitial fluid, saliva, a tissue sample, a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample.
Embodiments of the present disclosure also include a method of detecting an analyte in a sample comprising combining any of the compositions described above with a sample comprising a target analyte. In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from an analyte detection complex. In some embodiments, the method further comprises quantifying a bioluminescent signal generated from the analyte detection complex. In some embodiments, the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte. In some embodiments, one or more of the components of the composition exhibits enhanced stability within the composition compared to the component in solution alone.
The various embodiments of the compositions and formulations described above demonstrate enhanced stability, as demonstrated in the Examples and FIGS. For example, when produced as a dried formulation such as a lyocake, when dried onto a substrate or matrix (e.g., Whatman 903, Ahlstrom 237, and Ahlstrom 6613H; wells of a 96-well plate, nylon mesh), or when dried in various protein buffer formulations, with or without the luminogenic substrate, the compositions and formulations of the present disclosure exhibit enhanced stability when stored for a prolonged period of time. As provided herein, the compositions and formulations of the present disclosure are able to generate a luminescent signal in the presence of a target analyte after storage for extended periods of time. In some embodiments, the compositions and formulations of the present disclosure exhibit enhanced stability as compared to compositions and formulations that contain the same or similar components of a bioluminescent complex (e.g., complementary peptides/polypeptides, luminogenic substrates), but which are formulated without one or more of the other components of the formulation, and/or are not formulated according to the methods described herein.
In some embodiments, the compositions and formulations of the present disclosure exhibit enhanced stability for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 12 months, and up to 1 year. In some embodiments, the compositions and formulations of the present disclosure exhibit enhanced stability at temperatures ranging from about 0° C. to 65° C., from about 4° C. to 65° C., from about 10° C. to 65° C., from about 15° C. to 65° C., from about 15° C. to 65° C., from about 20° C. to 65° C., from about 25° C. to 65° C., from about 30° C. to 65° C., from about 35° C. to 65° C., from about 37° C. to 65° C., from about 40° C. to 65° C., from about 45° C. to 65° C., from about 50° C. to 65° C., from about 55° C. to 65° C., from about 60° C. to 65° C., from about 4° C. to 55° C., from about 10° C. to 50° C., from about 15° C. to 45° C., and from about 20° C. to 40° C.
Embodiments of the present disclosure include compositions, systems, assays, and methods for detecting one or more analytes in a sample. In accordance with these embodiments, described below are exemplary assays and devices for use with various embodiments herein. The following devices and assays should not be viewed as limiting the full scope of the systems, assays, and methods described herein.
a. Lateral Flow Assays
In certain embodiments, the present disclosure provides compositions and materials for conducting a lateral flow assay (e.g., a lateral flow immunoassay). Lateral flow assays are based on the principles of immunochromatography and can be used to detect, quantify, test, measure, and monitor a wide array of analytes, such as, but not limited to, analytes pertaining to monitoring ovulation, detecting/diagnosing infectious diseases/organisms, analyzing drugs of abuse, detecting/quantifying analytes important to human physiology, security screening, veterinary testing, agriculture applications, environmental testing, product quality evaluation, etc.
As shown in
In accordance with these embodiments, upon addition of a sample to the sample pad, the facilitation of lateral flow causes a target analyte within the sample to contact a first target analyte binding agent within the conjugate pad; subsequently, lateral flow causes the target analyte and the first target analyte binding agent to contact a second target analyte binding agent immobilized to a detection region of the analytical membrane. The presence and/or quantity of the target analyte is then determined based on detection of the analyte in the detection region (e.g., in the presence of a luminogenic substrate for the bioluminescent complex) and/or in comparison to the control.
In some embodiments, the above lateral flow systems make use of one or more NanoLuc®-based technologies (e.g., NanoBiT, NanoTrip, NanoBRET, etc.) for detection of the bound target analyte.
In an exemplary embodiment, as shown in
As shown in
In an alternative lateral flow embodiment to the one depicted in
In another exemplary alternative embodiment, a target analyte is an antibody generated in a subject in response to being exposed to an infectious disease/organism. The first target analyte binding agent includes a both a target analyte binding element that binds the antibody (e.g., a non-specific antibody binding agent (e.g., protein L), a target-specific (e.g., antibody) binding agent) and a first non-luminescent (NL) peptide tag (e.g., SEQ ID NO: 13 or 11 or variants thereof) capable of interacting with a second non-luminescent (NL) peptide (e.g., SEQ ID NO: 11 or 13 or variants thereof) and a non-luminescent (NL) polypeptide (e.g., SEQ ID NO: 12 or variants thereof) to generate a bioluminescent signal. The second target analyte binding agent includes a target analyte binding element that binds the antibody (e.g., a target-specific (e.g., antibody) binding agent, a non-specific antibody binding agent (e.g., protein L)) and a second NL peptide tag (e.g., SEQ ID NO: 11 or 13 or variants thereof). Formation of the bioluminescent complex in the presence of the NL polypeptide component (e.g., SEQ ID NO: 12 or variants thereof) and a luminogenic substrate in the detection region indicates the presence of the target analyte in the sample. The bioluminescent signal is detected and/or quantified to detect/quantity the antibody in the sample.
Additional alternatives to the exemplary embodiments set forth above are contemplated. For example, alternative binding agents, target analytes, detectable elements, order of the various components (e.g., non-specific binding agent/target-specific binding agent, target-specific binding agent/non-specific binding agent, target-specific binding agent/target-specific binding agent, etc.) are described herein and embodiments incorporating various combinations of the components are within the scope herein.
In some embodiments, a target analyte is not an antibody, but is instead a small molecule, peptide, protein, carbohydrate, lipid, etc. In some embodiments, the lateral flow assays and systems described above are configured (e.g., using one or more NanoLuc®-based technologies (e.g., NanoBiT, NanoTrip, NanoBRET, etc.)) for the detection of any such target analytes.
b. Solid Phase Assays
Embodiments of the present disclosure include compositions, assays, systems, devices, and methods for detecting one or more analytes in a sample. In accordance with these embodiments, the present disclosure provides compositions and materials for conducting a solid phase assay (e.g., a solid phase platform for conducting an immunoassay). Solid phase detection platforms are generally the simplest form of an immunoassay and can be used to detect, quantify, test, measure, and monitor a wide array of analytes such as, but not limited to, analytes pertaining to monitoring ovulation, detecting/diagnosing infectious diseases/organisms, analyzing drugs of abuse, detecting/quantifying analytes important to human physiology, veterinary testing, security screening, agriculture applications, environmental testing, and product quality evaluation. In contrast to lateral flow assays, solid phase detection platforms do not involve facilitating the flow of assay reagents across a membrane, but instead typically include a solid support to which components of the assay are attached or contained within (e.g., dipstick test or spot test).
As shown in
In accordance with these embodiments, a sample or a portion of a sample is applied to the detection and control regions of the solid phase assay platform such that a target analyte contacts a target analyte binding agent (220) conjugated to and/or within the detection region under conditions such that the binding event and/or the immobilization of the target analyte on the solid phase is detectable (e.g., a bioluminescent entity is immobilized, a bioluminescent complex is formed), thereby indicating the presence of the analyte in the sample.
In some embodiments, the solid phase assay platform includes a first target analyte binding agent (e.g., a target-specific binding agent (e.g., target-specific antibody, antigen for the target antibody, etc.)) immobilized on the solid phase. A second target analyte binding agent (e.g., a target-specific binding agent (e.g., target-specific antibody, antigen for the target antibody, etc.), a non-specific binding agent (e.g., protein L)) linked to a bioluminescent polypeptide (e.g., SEQ ID NO: 5 or variants thereof) is added to the solid phase with the sample (e.g., concurrently, sequentially, etc.). If both target analyte binding agent bind to the target analyte, the bioluminescent polypeptide becomes immobilized on the solid phase. Detection/quantification of bioluminescence on the solid phase (e.g., after a wash step) indicates the presence/amount of target analyte in the sample. In some cases, the first target analyte binding agent is conjugated to the detection region, and the second target analyte binding agent (attached to the bioluminescent polypeptide) is applied to the detection region with or without the sample. In some cases, the second target analyte binding agent is conjugated to the detection region, and the first target analyte binding agent (attached to the bioluminescent polypeptide) is applied to the detection region with or without the sample. In accordance with these embodiments, immobilization of bioluminescence at the detection region can be detected and/or quantified when in the presence of a luminogenic substrate (as described further below), thus indicating the presence (or absence) of the antibody in the sample.
In alternative embodiments, a solid phase assay platform utilizes a binary complementation approach, in which a bioluminescent complex is formed upon binding of two non-luminescent (NL) peptide/polypeptide components (e.g., NanoBiT system), to detect a target analyte. Multiple configurations of solid phase assays and systems utilizing a binary complementation approach are within the scope herein. For example, an exemplary system can include (i) a first target analyte binding agent linked to a first NL peptide or NL polypeptide (e.g., SEQ ID NOs: 9 or 10 or variants thereof) capable of interacting with high affinity with a second distinct NL polypeptide or NL peptide (e.g., SEQ ID NOs: 10 or 9 or variants thereof) to generate a bioluminescent signal, and (ii) a second target analyte binding agent linked to the complementary NL polypeptide or NL peptide, wherein the second target analyte binding agent is immobilized to the solid phase. Upon binding of the target analyte binding agents to the target analyte, a bioluminescent complex is formed on the solid phase and the bioluminescent signal is detectable/quantifiable, when in the presence of a luminogenic substrate (as described further below).
In other embodiments, a solid phase assay platform utilizes a tripartite complementation approach, in which a bioluminescent complex is formed upon binding of two non-luminescent (NL) peptide components and a non-luminescent (NL) polypeptide component (e.g., NanoTrip system), to detect a target analyte. In some embodiments, the solid phase assay platform includes: (i) a first target analyte binding agent comprising both a target analyte binding element (e.g., general or specific) and a NL peptide (e.g., SEQ ID NOs: 11 or 13) capable of forming a tripartite bioluminescent complex (e.g., NanoTrip complex), (ii) a second target analyte binding agent comprising both a target analyte binding element (e.g., specific) and a NL peptide (e.g., SEQ ID NOs: 11 or 13) capable of forming a tripartite bioluminescent complex (e.g., NanoTrip complex), (iii) a NL polypeptide component of the tripartite bioluminescent complex (e.g., NanoTrip complex), and (iv) a luminogenic substrate. In some cases, the first target analyte binding agent is conjugated to the detection region, and the second target analyte binding agent is applied to the detection region with or without the sample. In some cases, the second target analyte binding agent is conjugated to the detection region, and the first target analyte binding agent is applied to the detection region with or without the sample. Once the bioluminescent complex forms at the detection region, the bioluminescent signal is detected and/or quantified, thus indicating the presence (or absence) of the antibody in the sample.
In other embodiments, the solid phase assay platform includes (i) a first target analyte binding agent comprising a target analyte binding element and a NanoLuc®-based peptide or polypeptide, (ii) target analyte binding agent comprising a target analyte binding element and a fluorophore, and (iii) optionally the additional peptide/polypeptide components to form a bioluminescent complex (e.g., in embodiments in which the NanoLuc®-based peptide or polypeptide is not a bioluminescent polypeptide, e.g. non-luminescent), wherein upon binding of the first and second target analyte binding agents to a target analyte in a sample, in the presence of any additional components necessary for bioluminescence (e.g., luminogenic substrate, complementary components, etc.), emission from the NanoLuc®-based components (e.g., NanoLuc® protein or bioluminescent complex) excites the fluorophore (e.g., via BRET). In some cases, the first target analyte binding agent is conjugated to the detection region, and the second target analyte binding agent is applied to the detection region with or without the sample. In some cases, the second target analyte binding agent is conjugated to the detection region, and the first target analyte binding agent is applied to the detection region with or without the sample.
As shown in
In some embodiments, the solid phase platforms of the present disclosure can include a plurality of detection regions such as one or more wells of a microtiter plate, for example. In such embodiments, one or more distinct target analyte binding agents can be conjugated (e.g., coated) to wells of the microtiter plate along one or more of the other detection reagents required to carry out a particular bioluminescent assay (e.g., a second target analyte binding agent, a luminogenic substrate, assay buffer, etc.). In some embodiments, one or more of the other detection reagents (reagents not conjugated to the microtiter plate) required to carry out the assay can be added to the wells of the microtiter plate in the form of a lyophilized cake (lyocake) or tablet and reconstituted as part of the bioluminescent assay.
c. Solution Phase Assays
Embodiments of the present disclosure include compositions, assays, systems, devices, and methods for detecting one or more analytes in a sample. In accordance with these embodiments, the present disclosure provides compositions and materials for conducting a solution phase assay (e.g., a liquid-based format for conducting an immunoassay within a solution). Solution phase detection platforms can be used to detect, quantify, test, measure, and monitor a wide array of analytes such as, but not limited to, analytes pertaining to monitoring ovulation, detecting/diagnosing infectious diseases/organisms, analyzing drugs of abuse, detecting/quantifying analytes important to human physiology, veterinary testing, security screening, agriculture applications, environmental testing, and product quality evaluation. In contrast to lateral flow assays and solid phase detection platforms, solution phase detection platforms typically include a receptacle for the solution/liquid in which reactions involving the detection reagents take place, instead of conjugating one or more of the detection reagents to a solid support or membrane to facilitate detection.
For example, as shown in
In some embodiments, the solution phase assay platform includes a lyocake or tablet comprising one or more of a first target analyte binding agent (e.g., a target-specific binding agent (e.g., target-specific antibody, antigen for the target antibody, etc.)), a second target analyte binding agent (e.g., a target-specific binding agent (e.g., target-specific antibody, antigen for the target antibody, etc.), and a non-specific binding agent (e.g., protein L)) linked to a bioluminescent polypeptide (e.g., SEQ ID NO: 5 and variants thereof). Detection/quantification of bioluminescence in the solution indicates the presence/amount of target analyte in the sample.
In some embodiments, a solution phase assay platform utilizes a binary complementation approach, in which a bioluminescent complex is formed upon binding of two non-luminescent (NL) peptide/polypeptide components (e.g., NanoBiT system), to detect a target analyte. Multiple configurations of solution phase assays and systems utilizing a binary complementation approach are within the scope herein. For example, an exemplary system can include (i) a first target analyte binding agent linked to a first NL peptide or NL polypeptide (e.g., SEQ ID NOs: 9 or 10 or variants thereof) capable of interacting with high affinity with a second distinct NL polypeptide or NL peptide (e.g., SEQ ID NOs: 10 or 9 or variants thereof) to generate a bioluminescent signal, and (ii) a second target analyte binding agent linked to the complementary NL polypeptide or NL peptide. Upon binding of the target analyte binding agents to the target analyte, a bioluminescent complex is formed in the solution and the bioluminescent signal is detectable/quantifiable, when in the presence of a luminogenic substrate (as described further below).
In other embodiments, a solution phase assay platform utilizes a tripartite complementation approach, in which a bioluminescent complex is formed upon binding of two non-luminescent (NL) peptide components and a non-luminescent (NL) polypeptide component (e.g., NanoTrip system), to detect a target analyte. In some embodiments, the solution phase assay platform includes: (i) a first target analyte binding agent comprising both a target analyte binding element (e.g., general or specific) and a NL peptide (e.g., SEQ ID NOs: 11 or 13) capable of forming a tripartite bioluminescent complex (e.g., NanoTrip complex), (ii) a second target analyte binding agent comprising both a target analyte binding element (e.g., specific) and a NL peptide (e.g., SEQ ID NOs: 11 or 13) capable of forming a tripartite bioluminescent complex (e.g., NanoTrip complex), (iii) a NL polypeptide component of the tripartite bioluminescent complex (e.g., NanoTrip complex), and (iv) a luminogenic substrate. Once the bioluminescent complex forms in the solution, the bioluminescent signal is detected and/or quantified, thus indicating the presence (or absence) of the antibody in the sample.
In other embodiments, the solution phase assay platform includes (i) a first target analyte binding agent comprising a target analyte binding element and a NanoLuc®-based peptide or polypeptide, (ii) target analyte binding agent comprising a target analyte binding element and a fluorophore, and (iii) optionally the additional peptide/polypeptide components to form a bioluminescent complex (e.g., in embodiments in which the NanoLuc®-based peptide or polypeptide is not a bioluminescent polypeptide, e.g., non-luminescent), wherein upon binding of the first and second target analyte binding agents to a target analyte in a sample, in the presence of any additional components necessary for bioluminescence (e.g., luminogenic substrate, complementary components, etc.), emission from the NanoLuc®-based components (e.g., NanoLuc® protein or bioluminescent complex) excites the fluorophore (e.g., via BRET).
Solution phase platforms of the present disclosure can be configured to test for multiple different analytes (e.g., multiplexing), such as antibodies generated to distinct diseases/microorganisms in a single sample from a subject. In some embodiments, one or more of the detection reagents required to carry out a bioluminescent reaction to detect/quantify an analyte are present in one or more receptacles of a particular assay platform being used (e.g., individual wells of a 96-well plate), for example, as a lyocake or tablet that is to be reconstituted in a buffered solution. In other embodiments, one or more types of a sample solution are already present in the receptacles, and one or more lyocakes or tables are added to the receptacles and rehydrated to facilitate a bioluminescent reaction. In accordance with these embodiments, the solution phase platforms can include a plurality of receptacles comprising a distinct target analyte binding agent having distinct target analyte binding elements (e.g., distinct disease antigens).
d. Other Assays
Embodiments of the present disclosure include compositions, assays, systems, devices, and methods for detecting one or more analytes in a sample using other assay platforms known in the art. For example, target analytes can be detected and/or measured using the bioluminescent polypeptides and/or complexes described herein in the context of a microfluidic and/or chip-based assay. Because microfluidic systems integrate a wide variety of operations for manipulating fluids, such as chemical or biological samples, these systems are applicable to many different areas, such as biological and medical diagnostics. One type of microfluidic device is a microfluidic chip. Microfluidic chips may include micro-scale features (or micro-features), such as channels, valves, pumps, and/or reservoirs for storing fluids, for routing fluids to and from various locations on the chip, and/or for reacting fluidic reagents.
Microfluidic chips, or labs-on-a-chip (LOC), configured with bioluminescent polypeptides and/or complexes that include peptides and polypeptides capable of generating a bioluminescent signal in the presence of the target analyte offer increased flexibility for automation, integration, miniaturization, and multiplexing. For example, pathogen detection based on microfluidic chips use reaction chambers that are usually on the micro- or nano-scale, which allows devices to be miniaturized and portable; this is particularly advantageous for point-of-care testing. LOC technology allows for the integration of sample preparation, amplification, and signal detection, which reduces the time need to generate results. The high throughput and low consumption of sample and reagents make the technology flexible and relatively cost effective. Nucleic acid-based microfluidic pathogen detection for the detection of bacteria, viruses, and fungi that eliminates the need for PCR or real-time PCR for amplification is a distinct advantage of the bioluminescent complexes of the present disclosure.
Embodiments of the present disclosure also include methods of manufacturing an assay platform for use with bioluminescent peptides and polypeptides for target analyte detection. Although assay platforms may vary depending on various factors, such as the analyte being detected, the complexity of the sampling environment, and the diagnostic parameters, the compositions, materials and methods of the present disclosure can be applied to most currently available assay platforms, such as solid phase assays, lateral flow assays, and microfluidic-based assays.
a. Luminogenic Substrates
In some embodiments, methods of manufacturing assay platforms of the present disclosure include application of a luminogenic substrate. Luminogenic substrates, such as coelenterazine, and analogs and derivatives thereof, can decompose during storage thereby resulting in loss of the substrate before addition to or use in a biological assay. Such decomposition can be the result of instability of the luminogenic substrate in solution over time in a temperature-dependent manner. This decomposition results in waste of the luminogenic substrate and reduced sensitivity and reproducibility of luminescent measurements derived from biological assays that employed the decomposed luminogenic substrate.
Provided herein are compositions that include a luminogenic substrate, such as coelenterazine or an analog or derivative thereof. Exemplary coelenterazine analogs include coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, and JRW-1744.
In some embodiments, the substrate is coelenterazine, which has the following structure:
Exemplary coelenterazine analogs include coelenterazine-h (2-deoxycoelenterazine or 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one), coelenterazine-h-h (dideoxycoelenterazine or 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), furimazine (8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), JRW-0238 (8-benzyl-2-(furan-2-ylmethyl)-6-(3-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one), JRW-1404 (8-benzyl-6-(2-fluoro-3-hydroxyphenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one), JRW-1482 (. 6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one), JRW-1667 (6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one), JRW-1744 (6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-c]pyrazin-3(7H)-one), and JRW-1743 (6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-c]pyrazin-3(7H)-one), which have the following structures:
Additional exemplary coelenterazine analogs include coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-i, coelenterazine-icp, coelenterazine-v, 2-methyl coelenterazine, and the like. In some embodiments, the compound may be a coelenterazine analog described in WO 2003/040100; U.S. Pat. Pub. 2008/0248511 (e.g., paragraph [0086]); U.S. Pat. No. 8,669,103; WO 2012/061529; U.S. Pat. Pub. 2017/0233789; U.S. Pat. No. 9,924,073; U.S. Pat. Pub. 2018/0030059; U.S. Pat. No. 10,000,500; U.S. Pat. Pub. 2018/0155350; U.S. patent application Ser. No. 16/399,410 (PCT/US2019/029975); U.S. patent application Ser. No. 16/548,214 (PCT/US2019/047688); U.S. Pat. Pub. 2014/0227759; U.S. Pat. Nos. 9,840,730; 7,268,229; 7,537,912; 8,809,529; 9,139,836; 10,077,244; 9,487,520; 9,924,073; 9,938,564; 9,951,373; 10,280,447; 10,308,975; 10,428,075; the disclosures of which are incorporated by reference herein in their entireties. In some embodiments, coelenterazine analogs include pro-substrates such as, for example, those described in U.S. Pat. Pub. 2008/0248511; U.S. Pat. Pub. 2012/0707849; U.S. Pat. Pub. 2014/0099654; U.S. Pat. Nos. 9,487,520; 9,927,430; 10,316,070; herein incorporated by reference in their entireties. In some embodiments, the compound is furimazine. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744.
Provided herein are compositions that include a luminogenic substrate, such as coelenterazine or an analog or derivative thereof, and a polymer or a paper/fibrous substrate for the manufacture of bioluminescent target analyte detection platforms. Compositions that stabilize and/or enhance the reconstitution efficiency of luminogenic substrates such as coelenterazine or an analog or derivative thereof, are described in U.S. patent application Ser. No. 16/592,310 (PCT/US2019/054501); herein incorporated by reference in its entirety. In some embodiments, the composition stabilizes the compound against decomposition. In some embodiments, the composition stabilizes the compound against decomposition as compared to a composition that does not contain the polymer or paper/fibrous substrate. In some embodiments, the polymer or the paper/fibrous substrate reduces or suppresses the formation of one or more decomposition products from the compound. In some embodiments, the compositions enhance the reconstitution efficiency or reconstitution rate of the substrate.
Additionally, embodiments of the present disclosure include means for stabilizing (e.g., enhancing storage stability) the compositions described further herein. In some embodiments, enhancing the storage stability of the compositions provided herein includes methods and compositions for stabilizing a luminogenic substrate. The luminogenic substrate may be, but is not limited to, coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, a derivative thereof, an analog thereof, or any combination thereof. The compositions may include the luminogenic substrate, a thionucleoside, and an organic solvent. The composition may not include or contain a luminogenic enzyme. As provided in U.S. Pat. No. 9,676,997, which is herein incorporated by reference, a thionucleoside may be a compound of formula (I) or a tautomer thereof,
wherein
R1 is hydrogen, alkyl, substituted alkyl, alkyl-aryl, alkyl-heteroaryl, cycloalkyl, aryl, heteroaryl, carboxylic acid, ester, NRaRb, imine, hydroxyl, or oxo;
R2 is hydrogen, NRaRb, imine, alkyl, or aryl; and
Ra and Rb are each independently hydrogen, alkyl, or aryl.
In some embodiments, the compound of formula (I) may be ATT (6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one); 3-(4-Amino-5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid; tetrahydro-2-methyl-3-thioxo-1,2,4-triazine-5,6-dione; 4-((2-furylmethylene)amino)-3-mercapto-6-methyl-1,2,4-triazin-5(4H)-one; 6-benzyl-3-sulfanyl-1,2,4-triazin-5-ol; 4-amino-3-mercapto-6-methyl-1,2,4-triazin-5(4H)-one; 3-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid; (E)-6-methyl-4-((thiophen-2-ylmethylene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; (E)-6-methyl-4-((3-nitrobenzylidene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; (E)-4-((4-(diethylamino)benzylidene)amino)-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; ATCA ethyl ester; TAK-0021, TAK-0020, TAK-0018, TAK-0009, TAK-0014, TAK-0007, TAK-0008, TAK-0003, and TAK-0004, as provided in U.S. Pat. No. 9,676,997 (incorporated herein by reference); 3-thioxo-6-(trifluoromethyl)-3,4-dihydro-1,2,4-triazin-5(2H)-one; 6-cyclopropyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; 6-(hydroxymethyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-on; or any combinations thereof.
In some embodiments, a thionucleoside may stabilize the luminogenic substrate against decomposition over time, in the presence of light, in the absence of light, and/or at different temperatures. The thionucleoside may stabilize the luminogenic substrate against decomposition into one or more decomposition products over time, in the presence of light, in the absence of light, and/or at different temperatures. As such, inclusion of the thionucleoside in the compositions described further herein may stabilize the luminogenic substrate against decomposition by suppressing or reducing the formation of the one or more decomposition products as compared to a composition that does not include the thionucleoside. This, in turn, provides the capability of storing or incubating the luminogenic substrate for a period of time at a particular temperature, in the presence of light, and/or in the absence of light without significant decomposition of the luminogenic substrate before use of the luminogenic substrate in an assay. In accordance with these embodiments, the inclusion of a thionucleoside in the compositions described herein can enhance storage stability of the compositions. These embodiments also relate to methods for stabilizing the luminogenic substrate. Such a method may stabilize the luminogenic substrate against decomposition and/or suppress or reduce the formation of the one or more decomposition products. The method may include contacting the luminogenic substrate with an effective amount of the thionucleoside (e.g., 225 mM) in the presence of the organic solvent. This contacting step may include forming the composition described above.
In some embodiments, one or more of the non-luminescent (NL) peptide/polypeptide components that form the bioluminescent complexes described above can be included with or without a luminogenic substrate as part of a composition, such as a lyophilized powder. These compositions can be applied directly, with or without other components, to a portion of a detection platform, or they can be reconstituted as part of a separate solution that is applied to the detection platform.
Coelenterazine and analogs and derivatives thereof may suffer from challenges associated with their reconstitution into buffer systems used in many assays such as the bioluminogenic methods described herein. For example, coelenterazines, or analogs or derivatives thereof, such as furimazine, may dissolve slowly and/or inconsistently in buffer solutions (e.g., due to the heterogeneous microcrystalline nature of the solid material). While dissolution in organic solvent prior to dilution with buffer may provide faster and more consistent results, coelenterazine compounds may suffer from instability in organic solutions on storage, including both thermal instability and photo-instability. In some embodiments, the composition further comprises a polymer. As further described herein, the presence of the polymer may stabilize the compound against decomposition, and the presence of the polymer may improve the solubility of the compound in water or in aqueous solutions.
The polymer may be a naturally-occurring biopolymer or a synthetic polymer. In some embodiments, the polymer is a naturally-occurring biopolymer. Suitable naturally-occurring biopolymers are carbohydrates, including disaccharides (e.g., trehalose and maltose), and polysaccharides (e.g., pullulan, dextran, and cellulose). Mixtures of naturally-occurring biopolymers may also be used. In some embodiments, the polymer is pullulan, which is a polysaccharide that includes maltotriose repeating units. Maltotriose is a trisaccharide that includes three glucose units that are linked via α-1,4 glycosidic bonds. The maltotriose units within the pullulan polymer are linked to each other via α-1,6 glycosidic bonds.
In some embodiments, the polymer is a synthetic polymer. A synthetic polymer may be a homopolymer, copolymer, or block copolymer (e.g., diblock copolymer, triblock copolymer, etc.). Non-limiting examples of suitable polymers include, but are not limited to polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol) (PEG)), polyalkylene terephthalates (e.g., poly(ethylene terephthalate), etc.), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters (e.g., poly(vinyl acetate), etc.), polyvinyl halides (e.g., poly(vinyl chloride) (PVC), etc.), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses (e.g., alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, etc.), polymers of acrylic acids (“polyacrylic acids”) (e.g., poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polydioxanone and its copolymers (e.g., polyhydroxyalkanoates, polypropylene fumarate), polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, and mixtures and copolymers thereof.
In some embodiments, the composition further comprises a paper substrate. As further described herein, the presence of the paper substrate may stabilize the compound against decomposition, and the presence of the paper substrate may improve the solubility of the compound in aqueous solutions. Exemplary paper substrates include, but are not limited to, Whatman brand papers, (e.g., W-903 paper, FTA paper, FTA Elute paper, FTA DMPK paper, etc.), Ahlstrom papers (e.g., A-226 paper, etc.), M-TFN paper, FTA paper, FP705 paper, Bode DNA collection paper, nitrocellulose paper, nylon paper, cellulose paper, Dacron paper, cotton paper, and polyester papers, and combinations thereof.
In addition to the compound and the polymer and/or the paper substrate, the composition may include additional components such as buffers, surfactants, salts, proteins, or any combination thereof. For example, the composition may include a buffer such as a phosphate buffer, a borate buffer, an acetate buffer, or a citrate buffer, or other common buffers such as bicine, tricine, tris(hydroxymethyl)aminomethane (tris), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid (MES), or the like.
In some embodiments, the composition may include a surfactant. Exemplary surfactants include non-ionic surfactants, anionic surfactants, cationic surfactants, and zwitterionic surfactants. For example, the surfactant may be a non-ionic surfactant such as sorbitan 20.
In some embodiments, the composition may include a salt, such as sodium chloride, potassium chloride, magnesium chloride, or the like.
In some embodiments, the composition may include a protein. For example, the composition can include a carrier protein to prevent surface adsorption of luminogenic enzymes that may be added in downstream assays. In some embodiments, the protein may be bovine serum albumin (BSA).
In some embodiments, the composition may include a substance that reduces autoluminescence. In some embodiments, the substance is ATT (6-Aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, thiourea, and the like. In some embodiments, the substance is a thionucleoside disclosed in U.S. Pat. No. 9,676,997, herein incorporated by reference. In some embodiments, the substance is thiourea, which use for reducing autoluminescence is disclosed in U.S. Pat. Nos. 7,118,878; 7,078,181; and 7,108,996, herein incorporated by reference.
The composition may be in the form of a lyophilized powder. Such a composition can be prepared by drying a mixture of the components of the composition. For example, the composition can be prepared by dissolving the compound in a solvent (e.g., an organic solvent) to form a first solution, adding the polymer to the first solution to form a second solution, and then drying the second solution to provide the composition. In some embodiments, the drying step may comprise lyophilization. This may provide the composition in the form of a powder. In some embodiments, the drying step may comprise air-drying. This may provide the composition in the form of a malleable disk.
In some embodiments (e.g., those in which the composition includes a polymer rather than a paper substrate), the composition is in the form of a solution. When the composition is a solution, the composition may have a pH of about 5.5 to about 8.0, e.g., about 6.5 to about 7.5. In some embodiments, the composition has a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
b. Lateral Flow Components
In some embodiments, the present disclosure provides methods of manufacturing a lateral flow assay platform that includes a conjugate pad, an analytical membrane, a sample pad, and other components necessary for facilitating lateral flow across a membrane (e.g., an absorbent pad). For example, a conjugate pad can include at least one target analyte binding agent reversibly conjugated to the conjugate pad, such that the target analyte binding agent is able to be transferred from the conjugate pad to the analytical membrane when lateral flow is applied, whereupon the target analyte binding agent can bind a target analyte and form a bioluminescent complex. In some embodiments, the target analyte binding agent includes a target analyte binding element to facilitate binding to the target analyte, as well as a bioluminescent polypeptide or component of a bioluminescent complex, such as a bioluminescent polypeptide of SEQ ID NO: 5 (NanoLuc and variants thereof), a non-luminescent (NL) polypeptide of SEQ ID NO: 9 (LgBiT), an NL peptide of SEQ ID NO: 10 (SmBiT), an NL peptide of SEQ ID NO: 11 (HiBiT), an NL polypeptide of SEQ ID NO: 12 (LgTrip-3546), an NL peptide of SEQ ID NO: 13 (SmTrip), an NL peptide of SEQ ID NO: 14 (β9/β10 dipeptide), or variants thereof. In some embodiments, target analyte binding agent comprises a fluorophore capable of being activated by energy transfer (e.g., from a bioluminescent polypeptide or component of a bioluminescent complex).
In some embodiments, the conjugate pad comprises a first target analyte binding agent. In some embodiments, the first target analyte binding agent comprises a first target analyte binding element and a first bioluminescent polypeptide or a first component of a bioluminescent complex (e.g., NL peptide or NL polypeptide). In some embodiments, the target analyte binding agent is stored on or within the conjugate pad such that it remains with the conjugate pad until being displaced by lateral from through the device.
In some embodiments, the conjugate pad comprises a luminogenic substrate, such as coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, other coelenterazine analogs or derivatives, a pro-substrate, and/or other substrates (e.g., coelenterazine analog or derivative) described herein. In some embodiments, the luminogenic substrate is reversibly conjugated to the conjugate pad. In some embodiments, the luminogenic substrate is dried on or within the conjugate pad. In some embodiments, the luminogenic substrate is part of a composition comprising the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof (e.g., described in greater detail above and/or in U.S. Prov. Appln. Ser. No. 62/740,622. In some embodiment, the luminogenic substrate is applied as part of a composition or solution, such as a protein buffer. In some embodiment, the protein buffer includes 20 mM Na3PO4; 5% w/v BSA; 0.25% v/v Tween 20; 10% w/v sucrose. In some embodiments, luminogenic substrate is added to the protein buffer and dried for 1 hour at 37° C. onto a substrate or matrix (e.g., filter paper or membrane). In other embodiments, the luminogenic substrate is applied as a separate reagent as part of an assay method or system.
In some embodiments, the assay platform includes an analytical membrane comprising a detection region and a control region to facilitate the detection of the bioluminescent complex indicating target analyte detection. The detection region can include at least one target analyte binding agent immobilized to the detection region such that it will not be displaced by the application of lateral flow across the membrane. In some embodiments, the analytical membrane includes at least one target analyte binding agent. In some embodiments, the target analyte binding agent comprises a target analyte binding element and a bioluminescent polypeptide or a first component of a bioluminescent complex (e.g., NL peptide or NL polypeptide).
In some embodiments, the analytical membrane includes a plurality of detection regions with each detection region comprising a distinct target analyte binding agent comprising distinct target analyte binding elements (e.g., multiplexing capability).
In some embodiments, the analytical membrane comprises a luminogenic substrate, such as coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, other coelenterazine analogs or derivatives, a pro-substrate, or other substrates (e.g., coelenterazine analog or derivative) described herein. In some embodiments, the luminogenic substrate is reversibly conjugated to and/or contained on/within the analytical membrane, for example, as part of a composition comprising the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiment, the luminogenic substrate is applied as part of a composition or solution, such as a protein buffer. In some embodiment, the protein buffer includes 20 mM Na3PO4; 5% w/v BSA; 0.25% v/v Tween 20; 10% w/v sucrose. In some embodiments, the protein buffer includes 20 mM Na3PO4; 5% w/v BSA; 0.25% v/v Tween 20; 5% w/v pullulan. In some embodiments, the protein buffer includes 20 mM Na3PO4; 1-5% w/v BSA; 0.25% v/v Tween 20. In some embodiments, the protein buffer includes 20 mM Na3PO4; 1-5% w/v Prionex; 0.25% v/v Tween 20. In some embodiments, the protein buffer includes 20 mM Na3PO4; 1-5% w/v BSA, 5 mM ATT. In some embodiments, the protein buffer includes 20 mM Na3PO4; 1-5% v/v Prionex, 5 mM ATT. In some embodiments, the protein buffer includes 20 mM Na3PO4; 1-5% w/v BSA, 5 mM ATT, 5 mM ascorbate. In some embodiments, the protein buffer includes 20 mM Na3PO4; 1-5% w/v Prionex, 5 mM ATT, 5 mM ascorbate. In some embodiments, the protein buffer includes 20 mM Na3PO4; 1-5% w/v BSA, 5 mM ATT, 5 mM ascorbate. In some embodiments, the protein buffer includes; 1-5% w/v BSA, 5 mM ATT, 5 mM ascorbate. In some embodiments, luminogenic substrate is added to the protein buffer and dried for 1 hour at 37° C. onto a substrate or matrix (e.g., filter paper or membrane). In other embodiments, the luminogenic substrate is applied as a separate reagent as part of an assay method or system.
c. Solid Phase Components
In some embodiments, the present disclosure provides methods of manufacturing a solid phase detection platform (e.g., dipstick assay or spot test) that includes a detection region and a control region. In some embodiments, the detection region comprises at least one target analyte binding agent conjugated to the detection region. In some embodiments, the detection region comprises at least one target analyte binding agent that is not conjugated to the detection region. Such a non-conjugated binding agent may be added to the detection region (e.g., with the sample or as part of a detection reagent) or may reside on or within the detection region, without conjugation. In some embodiments, the non-conjugated binding agent comprises a target analyte binding element and bioluminescent polypeptide or component of a bioluminescent complex, such as a bioluminescent polypeptide of SEQ ID NO: 5 (NanoLuc and variants thereof), a non-luminescent (NL) polypeptide of SEQ ID NO: 9 (LgBiT), an NL peptide of SEQ ID NO: 10 (SmBiT), an NL peptide of SEQ ID NO: 11 (HiBiT), an NL polypeptide of SEQ ID NO: 12 (LgTrip-3546), an NL peptide of SEQ ID NO: 13 (SmTrip), an NL peptide of SEQ ID NO: 14 (β9/β10 dipeptide), or variants thereof.
In some embodiments, the solid phase detection platform includes a plurality of detection regions with each detection region comprising a distinct target analyte binding agent comprising distinct target analyte binding elements (e.g., multiplexing capability). In some embodiments, one or more distinct target analyte binding agents can be conjugated (e.g., coated) to wells of a microtiter plate, along one or more of the other detection reagents required to carry out a particular assay (e.g., a second target analyte binding agent, a luminogenic substrate, assay buffer, etc.). In other embodiments, the detection reagents can be applied as a separate reagent as part of an assay method or system (e.g., as part of a lyocake or tablet and reconstituted as part of the assay).
The detection platform can also include a luminogenic substrate, such as coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, other coelenterazine analogs or derivatives, a pro-substrate, or other substrates (e.g., coelenterazine analog or derivative) described herein. In some embodiments, the luminogenic substrate is reversibly conjugated to the detection region. In some embodiments, the luminogenic substrate is stably stored on or within the detection region. In some embodiments, the luminogenic substrate is part of a composition comprising the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the luminogenic substrate is applied as part of a composition or solution, such as a protein buffer, detection reagent, or with the sample. In some embodiments, the protein buffer includes 20 mM Na3PO4; 5% w/v BSA; 0.25% v/v Tween 20; 10% w/v sucrose. In some embodiments, luminogenic substrate is added to the protein buffer and dried for 1 hour at 37° C. onto a substrate or matrix (e.g., filter paper, membrane, individual wells of a microtiter plate). In other embodiments, the luminogenic substrate is applied as a separate reagent as part of an assay method or system (e.g., as part of a lyocake or tablet and reconstituted as part of the assay).
Embodiments of the present disclosure also include methods for producing a substrate or matrix for use in a bioluminescent assay. In accordance with these embodiments, the method includes generating a solution or liquid formulation containing at least one target analyte binding agent comprising a target analyte binding element and one of a polypeptide component of a bioluminescent complex or a peptide component of a bioluminescent complex. In some embodiments, the solution includes a protein buffer and at least one excipient, including but not limited to, a surfactant, a reducing agent, a salt, a radical scavenger, a chelating agent, a protein, or any combination thereof. In some embodiment, the solution includes a complementary peptide or polypeptide component of the bioluminescent complex, such that the target analyte binding agent and the complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of a target analyte. In some embodiments, the solution comprises a luminogenic substrate.
After generating the solution or liquid formulation, the method includes applying the solution to the surface of a substrate or matrix. In some embodiments, the substrate or matrix is W-903 paper, FTA paper, FTA Elute paper, FTA DMPK paper, Ahlstrom A-226 paper, M-TFN paper, FTA paper, FP705 paper, Bode DNA collection paper, nitrocellulose paper, nylon paper, cellulose paper, Dacron paper, cotton paper, and polyester papers, or combinations thereof. In other embodiments, the substrate or matrix is a mesh comprising plastic, nylon, metal, or combinations thereof.
Embodiments of the method also include drying the substrate or matrix after the solution has been applied to the substrate or matrix. In some embodiments, drying the substrate or matrix containing the solution comprises drying the substrate or matrix at a temperature from about 30° C. to 65° C., from about 30° C. to 60° C., from about 30° C. to 55° C., from about 30° C. to 50° C., from about 30° C. to 45° C., or from about 30° C. to 40° C. In some embodiments, the matrix or substrate is dried from about 15 mins to 8 hours, from about 30 mins to 7 hours, from about 45 mins to 6 hours, from about 1 hour to 5 hours, from about 2 hours to 4 hours, from about 30 mins to 2 hours, or from about 30 mins to 1 hour. In some embodiments, drying the substrate containing the solution comprises lyophilizing and/or freezing the substrate.
In some embodiments, the method includes drying the at least one target analyte binding agent and/or the complementary peptide or polypeptide component of the bioluminescent complex onto a first substrate, and drying the luminogenic substrate onto a second substrate. In some embodiments, the at least one target analyte binding agent and/or the complementary peptide or polypeptide component of the bioluminescent complex are dried onto a paper based substrate, and the luminogenic substrate is dried onto a mesh (see, e.g.,
In accordance with these embodiments, the substrate or matrix can be used in a bioluminescent assay to detect a target analyte. For example, a bioluminescent signal can be generated upon exposure of the substrate or matrix containing the solution to the target analyte. In some embodiments, the bioluminescent signal is proportional to the concentration of the target analyte. In some embodiments, the at least one target analyte binding agent and/or the complementary peptide or polypeptide component of the bioluminescent complex exhibit(s) enhanced stability when dried on the substrate, as described further herein.
d. Solution Phase Components
In some embodiments, the present disclosure provides methods of manufacturing a solution phase detection platform (as described herein) that includes one or more detection regions and control regions (e.g., wells of a 96-well microtiter plate). For example, as shown in
In some embodiments, one or more components of the bioluminescent complexes described herein can be added to a detection region and/or may already be present within a detection region, in the presence or absence of a sample. The detection reagents can then be reconstituted (e.g., rehydrated) as part of carrying out the detection of an analyte in the sample. In some embodiments, the detection reagent comprises a target analyte binding element and bioluminescent polypeptide or component of a bioluminescent complex, such as a bioluminescent polypeptide of SEQ ID NO: 5 (NanoLuc and variants thereof), a non-luminescent (NL) polypeptide of SEQ ID NO: 9 (LgBiT), an NL peptide of SEQ ID NO: 10 (SmBiT), an NL peptide of SEQ ID NO: 11 (HiBiT), an NL polypeptide of SEQ ID NO: 12 (LgTrip-3546), an NL peptide of SEQ ID NO: 13 (SmTrip), an NL peptide of SEQ ID NO: 14 (β9/β10 dipeptide), or variants thereof.
The solution phase detection platform can also include a luminogenic substrate, such as coelenterazine, coelenterazine-h, coelenterazine-h-h, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, other coelenterazine analogs or derivatives, a pro-substrate, or other substrates (e.g., coelenterazine analog or derivative) described herein. In some embodiments, the luminogenic substrate is part of a composition comprising the luminogenic substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and a combination of any thereof. In some embodiments, the luminogenic substrate is applied as part of a composition or solution, such as a protein buffer, detection reagent, or with the sample. In some embodiments, the luminogenic substrate is applied as a separate reagent as part of an assay method or system, and in other embodiments, it is part of a lyocake or tablet that includes one or more detection reagents.
Embodiments of the present disclosure find use in the detection/quantification of target analytes and include target analyte binding agents capable of binding to or interacting with a target analyte via a target analyte binding element. In some embodiments, target analyte binding agents include target analyte binding elements capable of binding a group or class of analytes (e.g., protein L binding generally to antibodies), such binding elements may be referred to herein as “non-specific” or the like; in other embodiments, target analyte binding agents include target analyte binding elements capable of binding a specific analyte (e.g., an antigen binding a monoclonal antibody), such binding elements may be referred to herein as “target specific” or the like.
In some embodiments, target analyte binding agents and corresponding target analyte binding elements are generated to detect one or more analytes associated with a disease state or environmental condition. Target analyte binding elements can be independently selected from the group consisting of an antibody (e.g., polyclonal, monoclonal, and/or recombinant), antibody fragment (e.g., Fab, Fab′, F(ab′)2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabodies, scFv, etc.), protein A, an Ig binding domain of protein A, protein G, an Ig binding domain of protein G, protein A/G, an Ig binding domain of protein A/G, protein L, a Ig binding domain of protein L, protein M, an Ig binding domain of protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein (e.g., either the analyte itself or a protein that binds to the analyte), and analyte binding domain(s) of proteins.
In some embodiments, target analyte binding elements comprise an antigen or epitope recognized by an antibody (the target analyte) such as an antibody generated by a subject in response to an immunogenic reaction to a pathogen, which can indicate that the subject is infected with the pathogen. In some embodiments, the target analyte is an antibody against Zika virus, Dengue virus, West Nile virus, Yellow Fever virus, and/or Chikungunya virus, and the target analyte binding element is an immunogenic epitope specifically recognized by the antibody. In some embodiments, the target analyte is an antibody against Hep A, B, C, D or E. In some embodiments, the target analyte is an antibody against Mumps, measles, Rubella, RSV, EBV, Herpes, Influenza, Varicella-Zoster, prenatal Zika, or parainfluenza type 1, 2, or 3. In some embodiments, the target analyte is an antibody against Arbovirus, HIV, prenatal Hepatitis, CMV, Hantavirus, polio virus, of parvovirus. In some embodiments, the target analyte is an antibody against Tick borne disease (e.g., Lyme disease). In some embodiments, the target analyte is an antibody against Bordetella pertussis, pneumococcus, chlamydia, streptococcus, M. pneumoniea, S. pneumonie, shigella producing bacteria, E. coli, Enterobacter, syphilis, gonorrhea. In some embodiments, the target analyte is an autoantibody against ANA, Cardiolipin, celiac disease, insulin, GAD65, IA-2, Reticulin, Thyroglobulin, RNP, cytoplasmic neutrophil, thyrptropin receptor, thyroperoxidase, platelet antibody, PLAR2, myocardial, GBM, tissue transglutaminase, or thyroid stimulating. In some embodiments, the target analyte is a toxin or an antibody against a toxin (e.g., diptheria, tetanus). In some embodiments, the target analyte is from a parasite or an antibody against a parasite (e.g., trichinella, trichinosis, Trypanosoma cruzi, Toxoplasma gondii). In some embodiments, the target analyte is a therapeutic biologic or an antibody against the therapeutic biologic (Vedolizumab, Adalimumab, infliximab, certilizumab, entanercept, Opdivo, Keytruda, ipilimumab, Ustekinumab, secukinumab, guselkumab, Tocilizumab, rituximab, panitumumab, trastuzumab, cetuximab, ofatumumab, eptratuzumab, abatacept, tofacitinib).
Other target analytes include known biomarkers associated with a pathogenic organism, such as a virus, bacterium, protozoa, prion, fungus, parasitic nematode, or other microorganism. Disease biomarkers can include markers of the pathogenic organism itself and/or markers of a subject's reaction to an infection by the pathogenic organism. Diseases that can be detected using the assays and methods of the present disclosure include any of the following: Acinetobacter infections (Acinetobacter baumannii), Actinomycosis (Actinomyces sraelii, Actinomyces gerencseriae and Propionibacterium propionicus), African sleeping sickness or African trypanosomiasis (Trypanosoma brucei), AIDS (HIV), Amebiasis (Entamoeba histolytica), Anaplasmosis (Anaplasma species), Angiostrongyliasis (Angiostrongylus), Anisakiasis (Anisakis), Anthrax (Bacillus anthracia), Arcanobacterium haemolyticum infection (Arcanobacterium haemolyticum), Argentine Teagan fever (Junin virus), Ascariasis (Ascaris lumbricoides), Aspergillosis (Aspergillus species), Astrovirus infection (Astroviridae family), Babesiosis (Babesia species), Bacillus cereus infection (Bacillus cereus), Bacterial pneumonia (multiple bacteria), Bacteroides infection (Bacteroides species), Balantidiasis (Balantidium coli), Bartonellosis (Bartonella), Baylisascaris infection (Baylisascaris species), BK virus infection (BK virus), Black Piedra (Piedraia hortae), Blastocystosis (Blastocystis species), Blastomycosis (Blastomyces dermatitidis), Bolivian hemorrhagic fever (Machupo virus), Brazilian hemorrhagic fever (Sabiá virus), Brucellosis (Brucella species), Bubonic plague (Yersinia Pestis), Burkholderia infection (usually Burkholderia cepacia and other Burkholderia species), Buruli ulcer (Mycobacterium ulcerans), Calicivirus infection (Caliciviridae family), Campylobacteriosis (Campylobacter species), Candidiasis (usually Candida albicans and other Candida species), Carrion's disease (Bartonella bacilliformis), Cat-scratch disease (Bartonella henselae), Cellulitis (usually Group A Streptococcus and Staphylococcus), Chagas Disease (Trypanosoma cruzi), Chancroid (Haemophilus ducreyi), Chickenpox (Varicella zoster virus or VZV), Chikungunya (Alphavirus), Chlamydia (Chlamydia trachomatis), Cholera (Vibrio cholerae), Chromoblastomycosis (usually Fonsecaea pedrosoi), Chytridiomycosis (Batrachochytrium dendrabatidis), Clonorchiasis (Clonorchis sinensis), Clostridium difficile colitis (Clostridium difficile), Coccidioidomycosis (Coccidioides immitis and Coccidioides posadasii), Colorado tick fever (Colorado tick fever virus or CTFV), Common cold (usually rhinoviruses and coronaviruses), Creutzfeldt-Jakob disease (PRNP), Crimean-Congo hemorrhagic fever (Crimean-Congo hemorrhagic fever virus), Cryptococcosis (Cryptococcus neoformans), Cryptosporidiosis (Cryptosporidium species), Cutaneous larva migrans (usually Ancylostoma braziliense; multiple other parasites), Cyclosporiasis (Cyclospora cayetanensis), Cysticercosis (Taenia solium), Cytomegalovirus infection (Cytomegalovirus), Dengue fever (Dengue viruses: DEN-1, DEN-2, DEN-3 and DEN-4), Desmodesmus infection (Green algae Desmodesmus armatus), Dientamoebiasis (Dientamoeba fragilis), Diphtheria (Corynebacterium diphtheriae), Diphyllobothriasis (Diphyllobothrium), Dracunculiasis (Dracunculus medinensis), Ebola hemorrhagic fever (Ebolavirus or EBOV), Echinococcosis (Echinococcus species), Ehrlichiosis (Ehrlichia species), Enterobiasis (Enterobius vermicularis), Enterococcus infection (Enterococcus species), Enterovirus infection (Enterovirus species), Epidemic typhus (Rickettsia prowazekii), Erythema infectiosum (Parvovirus B19), Exanthem subitum (Human herpesvirus 6 or HHV-6; Human herpesvirus 7 or HHV-7), Fasciolasis (Fasciola hepatica and Fasciola gigantica), Fasciolopsiasis (Fasciolopsis buski), Fatal familial insomnia (PRNP), Filariasis (Filarioidea superfamily), Fusobacterium infection (Fusobacterium species), Gas gangrene (usually Clostridium perfringens; other Clostridium species), Geotrichosis (Geotrichum candidum), Gerstmann-Sträussler-Scheinker syndrome (PRNP), Giardiasis (Giardia lamblia), Glanders (Burkholderia mallei), Gnathostomiasis (Gnathostoma spinigerum and Gnathostoma hispidum), Gonorrhea (Neisseria gonorrhoeae), Granuloma inguinale (Klebsiella granulomatis), Group A streptococcal infection (Streptococcus pyogenes), Group B streptococcal infection (Streptococcus agalactiae), Haemophilus influenzae infection (Haemophilus influenzae), Hand, foot and mouth disease (Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 or EV71), Hantavirus Pulmonary Syndrome (Sin Nombre virus), Heartland virus disease (Heartland virus), Helicobacter pylori infection (Helicobacter pylori), Hemolytic-uremic syndrome (Escherichia coli O157:H7, O111 and O104:H4), Hemorrhagic fever with renal syndrome (Bunyaviridae family), Hepatitis A (Hepatitis A virus), Hepatitis B (Hepatitis B virus), Hepatitis C (Hepatitis C virus), Hepatitis D (Hepatitis D Virus), Hepatitis E (Hepatitis E virus), Herpes simplex (Herpes simplex virus 1 and 2 (HSV-1 and HSV-2)), Histoplasmosis (Histoplasma capsulatum), Hookworm infection (Ancylostoma duodenale and Necator americanus), Human bocavirus infection (Human bocavirus or HBoV), Human ewingii ehrlichiosis (Ehrlichia ewingii), Human granulocytic anaplasmosis (Anaplasma phagocytophilum), Human metapneumovirus infection (Human metapneumovirus or hMPV), Human monocytic ehrlichiosis (Ehrlichia chaffeensis), Human papillomavirus (HPV) infection (Human papillomavirus or HPV), Human parainfluenza virus infection (Human parainfluenza viruses or HPIV), Hymenolepiasis (Hymenolepis nana and Hymenolepis diminuta), Epstein-Barr virus infectious mononucleosis (Epstein-Barr virus or EBV), Influenza (Orthomyxoviridae family), Isosporiasis (Isospora belli), Kingella kingae infection (Kingella kingae), Kuru (PRNP), Lassa fever (Lassa virus), Legionellosis (Legionella pneumophila), Legionellosis (Legionella pneumophila), Leishmaniasis (Leishmania species), Leprosy (Mycobacterium leprae and Mycobacterium lepromatosis), Leptospirosis (Leptospira species), Listeriosis (Listeria monocytogenes), Lyme disease (Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii), Lymphatic filariasis (Wuchereria bancrofti and Brugia malayi), Lymphocytic choriomeningitis (Lymphocytic choriomeningitis virus or LCMV), Malaria (Plasmodium species), Marburg hemorrhagic fever (Marburg virus), Measles (Measles virus), Middle East respiratory syndrome (Middle East respiratory syndrome coronavirus), Melioidosis (Burkholderia pseudomallei), Meningococcal disease (Neisseria meningitidis), Metagonimiasis (usually Metagonimus yokagawai), Microsporidiosis (Microsporidia phylum), Molluscum contagiosum (Molluscum contagiosum virus or MCV), Monkeypox (Monkeypox virus), Mumps (Mumps virus), Murine typhus (Rickettsia typhi), Mycoplasma pneumonia (Mycoplasma pneumoniae), Mycetoma (numerous species of bacteria (Actinomycetoma) and fungi (Eumycetoma)), Myiasis (parasitic dipterous fly larvae), Neonatal conjunctivitis (most commonly Chlamydia trachomatis and Neisseria gonorrhoeae), Norovirus (Norovirus), Nocardiosis (usually Nocardia asteroides and other Nocardia species), Onchocerciasis (Onchocerca volvulus), Opisthorchiasis (Opisthorchis viverrini and Opisthorchis felineus), Paracoccidioidomycosis (Paracoccidioides brasiliensis), Paragonimiasis (usually Paragonimus westermani and other Paragonimus species), Pasteurellosis (Pasteurella species), Pediculosis capitis (Pediculus humanus capitis), Pediculosis corporis (Pediculus humanus corporis), Pediculosis pubis (Phthirus pubis), Pertussis (Bordetella pertussis), Plague (Yersinia pestis), Pneumococcal infection (Streptococcus pneumoniae), Pneumocystis pneumonia (Pneumocystis jirovecii), Pneumonia (multiple causes), Poliomyelitis (Poliovirus), Prevotella infection (Prevotella species), Primary amoebic meningoencephalitis (usually Naegleria fowleri), Progressive multifocal leukoencephalopathy (JC virus), Psittacosis (Chlamydophila psittaci), Q fever (Coxiella burnetiid), Rabies (Rabies virus), Relapsing fever (Borrelia hermsii, Borrelia recurrentis, and other Borrelia species), Respiratory syncytial virus infection (Respiratory syncytial virus (RSV)), Rhinosporidiosis (Rhinosporidium seeberi), Rhinovirus infection (Rhinovirus), Rickettsial infection (Rickettsia species), Rickettsialpox (Rickettsia akari), Rift Valley fever (Rift Valley fever virus), Rocky Mountain spotted fever (Rickettsia rickettsia), Rotavirus infection (Rotavirus), Rubella (Rubella virus), Salmonellosis (Salmonella species), Severe Acute Respiratory Syndrome (SARS coronavirus), Scabies (Sarcoptes scabiei), Scarlet fever (Group A Streptococcus species), Schistosomiasis (Schistosoma species), Sepsis (multiple causes), Shigellosis (Shigella species), Shingles (Varicella zoster virus or VZV), Smallpox (Variola major or Variola minor), Sporotrichosis (Sporothrix schenckii), Staphylococcal food poisoning (Staphylococcus species), Staphylococcal infection (Staphylococcus species), Strongyloidiasis (Strongyloides stercoralis), Subacute sclerosing panencephalitis (Measles virus), Syphilis (Treponema pallidum), Taeniasis (Taenia species), Tetanus (Clostridium tetani), Tinea barbae (usually Trichophyton species), Tinea capitis (usually Trichophyton tonsurans), Tinea corporis (usually Trichophyton species), Tinea cruris (usually Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes), Tinea manum (Trichophyton rubrum), Tinea nigra (usually Hortaea werneckii), Tinea pedis (usually Trichophyton species), Tinea unguium (usually Trichophyton species), Tinea versicolor (Malassezia species), Toxocariasis (Toxocara canis or Toxocara cati), Toxocariasis (Toxocara canis or Toxocara cati), Toxoplasmosis (Toxoplasma gondii), Trachoma (Chlamydia trachomatis), Trichinosis (Trichinella spiralis), Trichomoniasis (Trichomonas vaginalis), Trichuriasis (Trichuris trichiura), Tuberculosis (usually Mycobacterium tuberculosis), Tularemia (Francisella tularensis), Typhoid fever (Salmonella enterica subsp. enterica, serovar typhi), Typhus fever (Rickettsia), Ureaplasma urealyticum infection (Ureaplasma urealyticum), Valley fever (Coccidioides immitis or Coccidioides posadasii), Venezuelan equine encephalitis (Venezuelan equine encephalitis virus), Venezuelan hemorrhagic fever (Guanarito virus), Vibrio vulnificus infection (Vibrio vulnificus), Vibrio parahaemolyticus enteritis (Vibrio parahaemolyticus), Viral pneumonia (multiple viruses), West Nile Fever (West Nile virus), White piedra (Trichosporon beigelii), Yersinia pseudotuberculosis infection (Yersinia pseudotuberculosis), Yersiniosis (Yersinia enterocolitica), Yellow fever (Yellow fever virus), Zygomycosis (Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthoramycosis)), and Zika fever (Zika virus).
Embodiments of the present disclosure include methods of detecting and/or quantifying a target analyte in a sample with an assay platform (e.g., solid phase detection platform or lateral flow assay) that uses bioluminescent polypeptides or bioluminescent complexes (and components thereof; e.g., non-luminescent peptide or polypeptides) for target analyte detection. Embodiments also include methods of diagnosing a disease state or evaluating an environmental condition based on detecting and/or quantifying a target analyte in a sample.
In some embodiments, a method of detecting an analyte in a sample includes using a lateral flow assay system or a solid phase detection platform as described herein. In accordance with these embodiments, the method includes applying a sample to a sample pad; facilitating flow of the sample from the sample pad to a conjugate pad, and then from the conjugate pad to a detection region and a control region on an analytical membrane. The method can include a first target analyte binding agent, a second target analyte binding agent, and a target analyte that form an analyte detection complex in the at least one detection region when the target analyte is detected in the sample. In some embodiments, methods comprise one or more steps of: sample addition, reagent (e.g., detection reagent) addition, washing, waiting, etc.
In some embodiments, the sample is a biological sample from a subject, such as blood, serum, plasma, urine, stool, cerebral spinal fluid, interstitial fluid, and saliva. In other embodiments, the sample is a sample from a natural or industrial environment, such as a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample. The method includes detecting the target analyte in the sample by detecting a bioluminescent signal generated from the analyte detection complex. In some embodiments, the target analyte in the sample is quantified based on the bioluminescent signal generated from the analyte detection complex. In some embodiments, the method includes diagnosing a subject from which the sample was obtained as having or not having a disease based on the detection of the analyte.
Some embodiments herein utilize competition between a labeled analyte and a target analyte in a sample to detect/quantify the target analyte in a sample. Exemplary embodiments comprise the use of (i) an analyte (e.g., identical or similar to the target analyte) labeled with detectable element described herein (e.g., NanoLuc®-based technology (e.g., NanoLuc, NanoBiT, NanoTrip, NanoBRET, or components (e.g., peptides, polypeptides, etc.) of variants thereof)), and (ii) a binding moiety for the target analyte (e.g., fused or linked to a second detectable element described herein (e.g., NanoLuc®-based technology (e.g., NanoLuc, NanoBiT, NanoTrip, NanoBRET, or components (e.g., peptides, polypeptides, etc.) of variants thereof)). In the absence of the target analyte from a sample, the detectable elements produce a detectable signal (e.g., via complementation between the detectable elements, via BRET, etc.) is produced by the system. When the system is exposed to a sample (e.g., biological sample, environmental sample, etc.), the bioluminescent signal is reduced if the target analyte is present in the sample (the labeled analyte will be competed out of the complex).
Various embodiments herein utilize such competition immunoassays for small molecule detection. In some embodiments, the target small molecule is a toxin (e.g., mycotoxin, etc.), metabolite (e.g., amino acid, glucose molecule, fatty acid, nucleotide, cholesterol, steroid, etc.), vitamin (e.g., vitamin A, vitamin B1, vitamin B2, Vitamin B3, vitamin B5, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin H or vitamin K, etc.), coenzyme or cofactor (e.g., coenzyme A, coenzyme B, coenzyme M, coenzyme Q, cytidine triphosphate, acetyl coenzyme A, reduced nicotinamide adenine dinucleodtide (NADH), nicotinamide adenine (NAD+), nucleotide adenosine monophosphoate, nucleotide adenosine triphosphate, glutathione, heme, lipoamide, molybdopterin, 3′-phosphoadenosine-5′-phsphosulfate, pyrroloquinoline quinone, tetrahydrobiopterin, etc.), biomarker or antigen (e.g., erythropoietin (EPO), ferritin, folic acid, hemoglobin, alkaline phosphatase, transferrin, apolipoprotein E, CK, CKMB, parathyroid hormone, insulin, cholesteryl ester transfer protein (CETP), cytokines, cytochrome c, apolipoprotein AI, apolipoprotein AII, apolipoprotein BI, apolipoprotein B-100, apolipoprotein B48, apolipoprotein CII, apolipoprotein CIII, apolipoprotein E, triglycerides, HD cholesterol, LDL cholesterol, lecithin cholesterol acyltransferase, paraxonase, alanine aminotransferase (ALT), asparate transferase (AST), CEA, HER-2, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA 125, CA 19.9, CA 15.3, leptin, prolactin, osteoponitin, CD 98, fascin, troponin I, CD20, HER2, CD33, EGFR, VEGFA, etc.), drug (cannabinoid (e.g., tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN), etc.), opioid (e.g., heroin, opium, fentanyl, etc.), stimulant (e.g., cocaine, amphetamine, methamphetamine, etc.), club drug (e.g., MDMA, flunitrazepam, gama-hydroxybutyrate, etc.), dissociative drug (e.g., ketamine, phencyclidine, salvia, dextromethorphan, etc.), hallucinogens (e.g., LSD, mescaline, psilocybin, etc.), etc.), explosive (e.g., 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), pentaerythritol tetranitrate (PETN), etc.), toxic chemical (e.g., tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), 2-(dimethylamino)ethyl N, N-dimethylphosphoramidofluroidate (GV), VE, VG, VM, VP, VR, VS, or VX nerve agent), etc.
In some embodiments, small molecule detection immunoassays, such as the one exemplified in Example 5 and the like, are performed in the solid phase, lateral flow, and other assays and devices described herein.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.
The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
Solid Phase Materials
As shows in
As shown in
Detecting Target Analytes with Bioluminescent Complexes
As shown in
As shown in
Additional tests of substrate and protein stability were performed and are depicted in
Additional tests of storage conditions were performed and are depicted in
Detecting Target Analytes in Complex Sampling Environments
Qualitative and Quantitative Assessment
These results demonstrate that the assays and methods of the present disclosure can include comparing levels of bioluminescence corresponding to target analyte detection with various control samples to facilitate rapid quantitative and qualitative assessment. For example, assay formats can include a plurality of control samples with varying concentrations of target analyte that can act as standards against which test samples can be assessed.
In accordance with these methods, a bioluminescent signal can be assessed both quantitatively and qualitatively using a high affinity dipeptide capable of forming a bioluminescent complex with LgBiT or LgTrip. The results shown in
Additionally,
Methods for assessing RLU activity for these experiments included imaging at day 6 for all at 25° C. and 37° C. (following the 4° C. time frame of 1 or 2 days); day 8 at 4° C. for LgTrip 3546; and day 9 for NanoLuc, LgBiT, and LgTrip 2098. Furimazine was tested at 50 μM and about 1.2 μM dipeptide was used for NanoBiT and NanoTrip experiments. All spots were placed into a plate with substrate reagents, images were captured with an iPhone and with an LAS4000 imaging system, then inserted into the plate reader. NanoGlo Live Cell Substrate cat #N205B (lot 189096) was used, along with assay buffer 1×PBS, pH 7.0).
Buffer Compositions
Experiments were also conducted to test short-term, or accelerated, stability of the complexes in different buffer compositions from 0 to 90 minutes. Methods included using about a 1.068 nM concentration of each protein absorbed and dried on Whatman 903 paper spots (⅛″). Protein samples were prepared and dried on paper spots with either protein buffer or PBS buffer (see each figure for specific buffer composition used). Stock concentrations included NanoLuc at 1000×(0.4 mg/mL), LgBiT-1672-11s-His at 20 μM, and LgTrip (3546) at 94 μM. Protein buffer was comprised of 20 mM Na3PO4, 5% w/v BSA, 0.25% v/v tween 20, 10% w/v sucrose. Luminescence activity was tested using the dipeptide added with furimazine in 100 μl assay buffer PBS, pH 7.0 (final [dipeptide]=1 nM; final [furimazine]=50 μM). Samples were read at time point 0 (fresh out of 4° C.), then placed into 60° C. and 25° C. for continued testing. A 1000×stock solution of NanoLuc was diluted 1:1000 in protein buffer (1 mL), or 10 μL of stock was diluted into 990 μL of protein buffer for a 1.068 nM stock (see each figure for specific buffer composition used). About 5 μL of each concentration was added to a paper spot for testing. For each protein tested (LgBiT and LgTrip), appropriate dilutions were made in each buffer to ensure that about 5 μL of 1.068 nM protein was used per spot. After all protein was added, the samples were dried at 35° C. for 1 hour, and 40 spots per condition and temperature were prepared.
For these experiments, the following solutions were prepared: (1) 5 μl mouse/antihuman into 995 μl Addition buffer (0.1 M PBS, pH 7.4); (2) 5 μl anti-mouse-NanoLuc in 995 μl Addition buffer (0.1 M PBS, pH 7.4); (3) 5 μl mouse/antihuman in protein buffer (20 mM Na3PO4, 5% w/v BSA, 0.25% v/v tween 20, 10% w/v sucrose); and (4) 5 μl anti-mouse-NanoLuc in 995 μl protein buffer (20 mM Na3PO4, 5% w/v BSA, 0.25% v/v tween 20, 10% w/v sucrose).). About 0.5 ml of solution (1) was loaded into an airbrush and applied to the left side of a nitrocellulose strip (Strip 1 and 2). The strips were allowed to dry either at RT or at 37° C. for 1 hour. About 0.5 ml of solution (2) for was applied to the entire surface of strip 1 and strip 2 and allowed to dry at RT or at 37° C.; forming condition 1 and 2, respectively. About 0.5 ml of solution (3) was loaded into an airbrush and applied to the left side of a nitrocellulose strip (Strip 3 and 4). The strip was allowed to dry either at RT or at 37° C. for 1 hour. About 0.5 ml of solution (4) for was applied to the entire surface of strip 3 and strip 4 and allow to dry at RT or 37° C.; forming condition 3 and 4, respectively. For imaging, a 1×solution of substrate was prepared (4 mls PBS+1 ml Nano-Glo LCS Dilution Buffer+50 ul Nano-Glo Live Cell Substrate) and overlaid on each strip with 1 ml of substrate solution; imaging began immediately thereafter.
These data demonstrate that buffer formulations are important for activity in lateral flow membranes. In conditions 1-4, where protein was just applied to the membrane in PBS, very little to no light was observed when the membranes were exposed to freshly prepared Nano-Glo Live Cell substrate. In contrast, protein that was prepared with a loading buffer that contained additional components such as Na3PO4, BSA, Tween 20, and sucrose showed considerable light output. This suggests that the particular loading buffer used to add the protein to the surface of the membrane is important for stability and function (
Lateral Flow Assay Components
Experiments were conducted to test different membrane blocking agents and assay running buffers to facilitate proper movement of proteins and targets during a lateral flow assay. Four strips were used, and the design of each (with or without sucrose and blocking agent) is shown in the schematic below the far left image of
The blocking buffer was comprised of 1% w/v polyvinyl alcohol in 20 mM tris, pH 7.4. Conjugation pre-treatment included 30% sucrose w/v in DI water. The conjugation pad was Ahlstrom grade 8950 (chopped glass with binder, 50 g/m2), and the membrane was nitrocellulose. For blocking, the membrane was soaked in blocking buffer for 30 min at RT, and subsequently remove from buffer, washed with DI water, and dried for 30 min at 35° C. For secondary pre-treatment, sucrose solution was applied to the membrane pad near where conjugation reagent (substrate) will be applied. The membrane was dried for lhr at 35° C. To prepare the proteins, about 5 μL anti-mouse-NanoLuc was added to 995 μl protein buffer. About 1 ml of protein solution was placed into an airbrush and a light coating was applied to the conjugation pad. This was allowed to dry for 1 hr at 35° C. Strips were then assembled on backing card. Additionally, for
Experiments were also conducted to assess different membranes and membrane properties within the context of a lateral flow assay such as the effects of membrane properties on absorption and capillary action.
Running buffer was comprised of 5×LCS dilution buffer+5×LCS—diluted to 1× in PBS. Membranes were pre-treated by applying 30% sucrose solution to the membrane, covering ˜1.5 cm of the bottom of the strip, the allowed to dry at 35° C. for 1 hour. Proteins were prepared by adding about 5 μL anti-mouse-NanoLuc in 995 μL protein buffer. About 1 mL of protein solution was added to an airbrush, which was used to lightly coat conjugation pad. This was allowed to dry at 35° C. for 1 hour. The negative control for these experiments contained protein buffer without protein, which was applied with an airbrush in the same manner as the test conditions. Strips were assembled on backing card. The conjugation pad, sample pad, and wicking pad were cut to be 2 cm×1 cm. The sample pad and conjugation pad were overlapped by ˜1.8 cm. The total dimensions of the strip were about 6 cm×1 cm.
An imaging program was created to capture 5 sec exposure images every 30 seconds for a total of about 10 minutes. Imaging was repeated if it appeared that there was still NanoLuc flowing across the membrane. Images were stacked into movies using ImageJ, and the final images included in
These results suggest that strips 4 and 6 (boxed in
Bioluminescent Complex Formation
Experiments were conducted to evaluate bioluminescent complex formation in the presence of various reagents on membrane and filter paper. Experiments were designed and conducted according to the schematic below, which shows the four different conditions tested.
For these experiments, 2.5 μL of HaloTag-HiBiT was added to 498 μL protein buffer. About 5 μL of this solution was spotted on both the membrane and filter paper in quadrants 1, 3, and 4 (see above schematic) and allowed to dry at 37° C. for 1 hour. About 2.5 μL of ATG-1672-11S-6His was diluted in 498 μL of protein buffer, and about 5 μL was spotted directly onto nitrocellulose membrane and filter paper in quadrants 2, 3, and 4 (see above schematic). Membranes were allowed to dry at RT for 1 hour. Furimazine was prepared as a 5 mM stock solution in EtOH. About 5 μL of this solution was spotted onto both the membrane, and the filter paper in quadrants 1, 2, and 3 and immediately placed under high vacuum for 15 minutes. About 2.5 μL of stock protein (20 μM) was diluted in 498 μL of NanoGLO buffer, which does not contain substrate. About 5 μL was added to the quadrant indicated above and subsequently read in a luminometer.
These results suggest that filter paper may provide an increased signal as compared to the membrane. Also, the conditions present in quadrant 4 did not produce detectable luminescence, which could indicate that complex formation was impeded by one or more of the other reagents present.
Experiments were conducted to assess the effects of increased substrate concentration on complex formation.
Spiking in additional substrate (furimazine) in an excess of liquid volume showed that signal returns, suggesting that as components start to move within the additional fluid, more complexes may be forming due to their increased mobility. This experiment also indicates that the enzyme retains activity with substrate concentration being the limiting factor that can be remedied by the addition of excess substrate.
Spot Tests with LgTrip and Substrate
Experiments were conducted to assess the feasibility of an “all-in-one” spot by first testing paper matrix containing LgTrip 3546 and furimazine to which an analyte-of-interest can be added (e.g., dipeptide).
To prepare the spots, a vial containing 200 μL of 5 μM LgTrip 3546, 5 mM ATT, and 5 mM ascorbic acid was prepared. About 5 μL of this solution was added to each spot, and the spots were then allowed to dry at 35° C. for 1 hour. After drying, 1 mM stock of furimazine in ethanol was prepared. About 5 μL of this solution was added to each spot and allowed to dry at 35° C. for an additional 30 minutes. For luminescent measurements, at the time of testing, 1.2 mM dipeptide stock in water was serial diluted down to 1e−10 M in PBS, pH 7.0. About 100 μL of each dipeptide stock was added to a 96-well plate containing a spot and kinetic measurements were started immediately.
These data demonstrate that a stable, concentration dependent response was observed with the addition of the dipeptide (
Lyophilized Cake Containing LgTrip and Substrate
These data demonstrate that a stable, concentration dependent bioluminescent response was observed with the addition of the dipeptide (
Protein Buffer Formulations
For
For these experiments, Whatman 903 protein saver spot cards were used with the following protein buffer formulations:
For NanoLuc, a 1000× stock solution was diluted 1:1000 in protein buffer (1 mL). For a 1.068 nM stock solution, 3 μl was diluted into 297 μl of protein buffer. About 5 μL of each concentration was spotted on the filter paper. For LgBiT-1672-11s-His, 5 μL of 1.068 nM protein per spot was used. About 104, was diluted in 990 μL protein buffer for a 2e−7M stock. About 100 μL of a 100 nM protein solution was then prepared, and about 10 μL stock was diluted into 990 μL protein buffer for 1 nM stock. About 5 μL of each concentration was spotted onto filter paper. For LgTrip 3546, about 5 μL of 1.068 nM protein was used per spot. About 1.1 of LgBiT-1672 stock was diluted into 998.94 μL protein buffer. About 3 μL stock was diluted into 297 μL protein buffer. About 54, of each concentration was spotted onto filter paper. After all protein was added, the samples were dried at 30° C. for about 1 hour. About 40 spots were made for each condition (see above schematic diagram). Spots were tested on day 0 for a baseline and then placed at 60° C. and tested 6 days later. RLU activity was tested by addition of 1 nM of high affinity dipeptide+50 μM live cell substrate in PBS, pH 7.0.
Experiments were also conducted to assess luminescent background levels in the various protein buffer compositions described above.
In
Lateral Flow Assays
For
The materials used for these experiments included a Conjugation pad (Ahlstrom grade 8950, chopped glass with binder, 50 g/m2), a Sample Pad (Cellose glass fiber CFSP203000 (Millipore)), an Absorption pad (Cotton linters, grade 238 (Ahlstrom)), a Membrane (nitrocellulose Hi-Flow Plus HFC07502 (Millipore), #6 from strip-test 2), and Running buffer (5×LCS dilution buffer+5×LCS diluted to 1× in PBS). Membranes were prepared by applying 30% sucrose solution to the membrane covering about 1.5 cm of the bottom of the strip. The membrane was allowed to dry at 35° C. for 1 hour. Strips were initially cut to be 4.5 cm×1 cm.
Protein preparations were carried out according to the conditions below:
Strips were assembled on backing card with conjugation pad, sample pad, and wicking pad cut to 1 cm×1 cm. Once strips were assembled, they were cut in half lengthwise to a final dimension of 4.5 cm×0.5 cm. For imaging analysis, about 250 μl 1×LCS buffer+LCS was diluted in PBS. Images were captured at 5 sec exposures with 5 sec wait time in between images; representative images are compilation images from corresponding movies taken across total exposure time (movies can be made available upon request). Total read time was 2:40 minutes.
Fumonisin Detection
Experiments were conducted during development of embodiments herein to demonstrate the use of NanoLuc®-based technologies in a competition-type immunoassay for the detection of a fumonisin B1, an exemplary small molecule toxin. Such assays can be performed in the devices and systems described herein, and with other small molecule targets and target analytes.
In an exemplary assay, tracers were generated by tethering fumonisin B1 to a NLpeptide tag (e.g., a peptide tag comprising SEQ ID NO: 10) via a biotin/streptavidin linkage, via a HaloTag linkage, or directly (
Lyophilized Cake Containing LgBiT and Substrate
For luminescent measurements, at time of testing, 1.2 mM dipeptide stock in water was serial diluted down to 1e−10 M in PBS, pH 7.0. 100 μl of each dipeptide stock was added to a lyophilized vial containing LgBiT and substrate, pipetted briefly to mix, and then placed into a 96-well plate and kinetic measurements were started immediately.
These data demonstrate that a stable, concentration dependent bioluminescent response was observed with the addition of the dipeptide. This experiment highlights that a solid format containing LgBiT and substrate can be made and then reconstituted in aqueous media containing a potential analyte of interest (e.g., dipeptide).
Substrate and LgTrip 3546 or LgBiT Lyophilization
Conditions 3 (LgTrip 3546/substrate) and 4 (LgBiT/substrate) were prepared as described above: 2.5% w/v pullulan in water containing 5 mM ATT and 5 mM ascorbic acid was prepared (solution 1, pH 6.5). Solution 1 was then aliquoted out into 45 μl volumes into each well of the plate. About 2.6 μl of 95 μM LgTrip 3546 protein or 5 μl of 20 μM LgBiT protein was added to each vial and pipetted to mix. Approximately 5 μl of 10 mM furimazine in ethanol was then added to each well forming condition 3 and 4 respectively. The plate was then placed in a cooler with dry ice to freeze for 1 hour, followed by lyophilization overnight.
For luminescent measurements, at time of testing, 1.2 mM dipeptide stock in water was serial diluted down to 1e−9M in PBS, pH 7.0 (
This data demonstrates that a lyocake containing either LgBiT or LgTrip 3546 and substrate can be prepared directly within a 96-well plate and reconstituted in the presence of an analyte of interest (dipeptide) leading to stable and robust signal.
Paper Based all-in-One Analyte Detection Systems
Experiments were conducted to test the efficacy of paper-based detection platforms containing NanoBiT (
Similar experiments were performed, as shown in
These experiments show that it is possible to build and all-in-one, paper-based bioluminescent assay platforms for the detection of an analyte-of-interest using both NanoBiT and NanoTrip complementation systems. In addition, these experiments demonstrate that it is possible to quantify the amount of analyte present in the sample matrix based on a change in overall light output. Increasing the concentration of the analyte-of-interest (i.e. Remicade) led to a proportional increase in the bioluminescent signal (the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte).
Lyocake Based all-in-One Analyte Detection Systems
Experiments were also conducted to test the efficacy of lyocake-based detection platforms containing NanoBiT (
As shown in
In
In the condition where the spots were exposed to assay buffer containing 1 nM Remicade, there was an increase in overall light output compared to the blank condition, which contained no Remicade. An increase in signal was observed as the concentration of Remicade increased to 100 nM. These experiments show that it is possible to build and all-in-one lyocake-based, bioluminescent-based assay platforms for the detection of an analyte-of-interest using both NanoBiT and NanoTrip complementation systems. In addition, these experiments demonstrate that it is possible to quantify the amount of analyte present in the sample matrix based on a change in overall light output. Increasing the concentration of the analyte-of-interest (i.e. Remicade) led to a proportional increase in the bioluminescent signal (the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte).
Mesh-Based Systems to Separate Substrate from Bioluminescent Complexes for Analyte Detection
Experiments were conducted to investigate the conditions required to generate a bioluminescent signal when peptide and polypeptide components of the bioluminescent complexes provided herein were produced in a format that does not include the substrate. For example, in one embodiment, an amount of a solution (e.g., containing an analyte-of-interest) is added to a mesh or matrix that has the luminogenic substrate adhered (“caked”) to it. Addition of the solution acts to reconstitute the substrate on the mesh, and this solution subsequently interacts with the surface of paper containing the dried down peptides and polypeptides of the bioluminescent complexes of the present disclosure, thus generating a bioluminescent signal (
As shown in
Experiments were also conducted using LgTrip 3546 bioluminescent components with the mesh-based format. The master mix, which was used to generate the paper spots containing the bioluminescent peptide/polypeptide components, included: 5% w/v BSA, 5 mM ATT, 5 mM ascorbate, 100 nM LgTrip 3546, at pH 6.5. About 10-20 μl of the master mix was added to the spots and then dried at about 35° C. for about 1 hour. To generate the mesh containing the substrate, a solution of about 0.75% pullulan in water was prepared. About 450 μl of this solution was added to a plastic snap-cap vial. About 50 μl of 10 mM furimazine in EtOH was added to the vial and pipetted to mix. About 25 μl of this solution was added to the top of the mesh-spots. The mesh spots were then frozen on dry-ice, and lyophilized overnight. At the time of testing, dipeptide ranging from 100 nM to 0.1 nM was prepared in PBS. The spots were placed in wells, and the screen containing the substrate was placed on the surface of the spots. About 10 μl of the solutions containing each concentration of peptide was added to the surface of the screen and RLU's were recorded (
Experiments were also conducted using LgTrip 3546 bioluminescent components with the mesh-based format and by forming a pullulan film. The master mix, which was used to generate the paper spots containing the bioluminescent peptide/polypeptide components, included: 5% w/v BSA, 5 mM ATT, 5 mM ascorbate, 100 nM LgTrip 3546, at pH 6.5. About 10-20 μl of the master mix was added to the spots and then dried at about 35° C. for about 1 hour. To generate the mesh containing the substrate, a solution of about 2.0% pullulan in water was prepared. About 450 μl of this solution was added to a plastic snap-cap vial. About 50 μl of 10 mM furimazine in EtOH was added to the vial and pipetted to mix. About 25 μl of this solution was added to the top of the mesh-spots. The spots were then allowed to dry under ambient conditions, in the dark, overnight. This method resulted in the formation of a pullulan film that filled the holes of the mesh. At the time of testing, dipeptide ranging from 100 nM to 0.1 nM was prepared in PBS. The spots were placed in wells, and the screen containing the substrate was placed on the surface of the spots. About 10 μl of the solutions containing each concentration of peptide was added to the surface of the screen and RLU's were recorded (
These experiments show that it is feasible to detect bioluminescent signal in a mesh-based format in which the peptide/polypeptide components are separate from the substrate. In addition, in the context of this format, these experiments demonstrate that increasing the concentration of the analyte-of-interest (i.e. dipeptide) leads to a proportional increase in the bioluminescent signal (the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte).
Testing Different Formulated, Lyophilized Substrates for Cake Appearance, Reconstituted Kinetic Activity Performance, and Accelerated Thermal Stability
To evaluate the potential application of lyophilization for preservation of the furimazine substrate, formulations containing furimazine were prepared. The 20× stock formulations were as follows:
Condition 1: 100 μM furimazine in ethanol, 5 mM azothiothymine, 5 mM ascorbic acid, 2.5% pullulan w/v, ddH20 (Millipore);
Condition 3: 100 μM furimazine in ethanol, 5 mM azothiothymine, 5 mM ascorbic acid, 2.5% pullulan w/v, 20 mM HEPES buffer (pH 8.0), 90 mM glycine, 20 mM histidine, 25 mg/ml sucrose, 0.01% polysorbate 80;
Condition 5: 40 μM furimazine in 85% ethanol+15% glycol, 200 mM MES buffer (pH 6.0), 200 mM hydroxyproyl beta cyclodextrin (m.w. 1396 Da), 600 mM sodium ascorbate, 2.5% pullulan w/v; and
Condition 7: 20 μM furimazine in ethanol, 200 mM MES buffer (pH 6.0), 200 mM hydroxyproyl beta cyclodextrin (m.w. 1396 Da), 600 mM sodium ascorbate, 2.5% pullulan w/v.
One mL aliquots of 20× stock solution was dispensed into 10 mL amber glass vials, and a runner stopper was partially inserted into the vial. Vials were loaded into a lyophilizer (Virtis Genesis 12EL lyophilizer) with shelves pre-chilled to 4.7° C. Product then underwent a freezing step with a shelf temperature of −50° C. for 2 hr after which time the condenser step started. During the run, the condenser temperature ran between −5° C. and −87° C. A vacuum pull down ran next at the pressure set-points of 75 and 200 mTorr. Sublimation lasted ˜7.5 hr, and desorption lasted ˜16.1 hr. At the end of the lyophilization process, the vials were back-filled with nitrogen and sealed with fully inserted stoppers at ˜600 Torr of pressure.
Vials were stored at 25° C. or 60° C. and tested at various timepoints post-lyophilization. For activity-based assays, furimazine cakes were reconstituted with 10 mL of PBS containing 0.01% BSA. The vials were shaken manually and allowed to equilibrate at room temperature for 5 minutes. Fifty μl of the reconstituted substrate was added to 50 μl of 1 ng/mL purified NANOLUC enzyme (Promega) that was reconstituted in the same BSA buffer (final [NanoLuc]=0.5 ng/ml). The controls used were the NANOGLO Live Cell Substrate (Promega Cat. N205) or NANOGLO substrate (Promega Cat. N113) according to manufacturer's protocol, but were diluted into PBS containing 0.01% BSA instead of the dilution buffer provided in the kit (Promega). Assays were performed in solid, white, nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega) collecting total luminescence using kinetic or endpoint reads, depending on the experiment. For analysis of absolute [furimazine], reconstituted samples were analyzed on HPLC for absorbance spectra at wavelength 245 nm and the absolute amount remaining from day 0 was plotted.
The appearance of the lyophilized cakes resulting from these formulations are displayed in
Accelerated thermal stability studies indicated no loss of activity for 3 months for the formulated and lyophilized furimazine for Condition 1, which in stark contrast to the furimazine stored in organic solvent, which lost all activity in about 10 days when stored at this elevated temperature (
Development of a Solution-Based, Homogeneous Human Interleukin-6 Tripartite Immunoassay Using HaloTag-Peptide Fusions to Chemically Conjugate Monoclonal Antibody Pairs
The basic principle of the homogeneous NanoLuc tripartite (NanoTrip) immunoassay is depicted in
Genetic fusions containing the SmTrip9 variants (SmTrip9 Pep521; SEQ ID NO: 16) or SmTrip10 variants (SmTrip10 Pep289 or VSHiBiT; SEQ ID NO: 17 separated by either a 2× or 3X Gly-Ser-Ser-Gly linker to the amino terminus of HaloTag was achieved using the pFN29A HIS6HaloTag T7 Flexi Vector (Promega). Glycerol stocks of E. coli expressing HisTag-HaloTag fusion protein was used to inoculate 50 mL starter cultures, which were grown overnight at 37° C. in LB media containing 25 ug/ml kanamycin. Starter cultures were diluted 1:100 into 500 mL fresh LB media containing 25 ug/mL kanamycin, 0.12% glucose, and 0.2% rhamnose. Cultures were grown for 22-24 h at 25° C. Cells were pelleted by centrifugation (10,000 rpm) for 30 min at 4° C. and re-suspended in 50 mL PBS. 1 mL protease inhibitor cocktail (Promega), 0.5 mL RQ1 DNase (Promega), and 0.5 mL of 10 mg/mL lysozyme (Sigma) were added, and the cell suspension was incubated on ice with mild agitation for 1 h. Cells were lysed by sonication at 15% power at 5 s intervals for 1.5 min (3 min total) and subsequently centrifuged at 10,000 rpm for 30 min at 4° C. Supernatant was collected, and protein purified using HisTag columns (GE) following manufacturer's recommended protocol. Protein was eluted using 500 mM imidazole, dialyzed in PBS, characterized using SDS-PAGE gel and was >95% pure. Proteins were stored in 50% glycerol at −20° C.
To chemically conjugate the antibodies to the HaloTag-peptide fusion proteins, antibodies were buffered exchanged 2× into 10 mM sodium bicarbonate buffer (pH 8.5) using Zeba spin desalting columns (ThermoFisher). Antibodies were then primed with 200 μM amine-reactive HaloTag Succinimidyl Ester (04) Ligand (Promega) for 2 hr shaking at 1000 rpm at 22° C. Unreacted ligand was removed with two passes through Zeba spin columns in PBS buffer. Then, antibodies were covalently labeled with 30 μM of the HaloTag fusion protein overnight at 4° C. while shaking. Excess unreacted HaloTag fusion protein was removed using HaloLink Resin (Promega). Non-denaturing SDS-PAGE gel was used to characterize the conjugated antibodies. Mouse anti-human IL-6 monoclonal antibodies used in the human IL-6 immunoassay were clone 5IL6 (Thermo cat #M620) and clone 505E 9A12 A3 (Thermo cat #AHC0662). SDS-PAGE gels were performed on the labeled antibodies and it was determined that each antibody was labeled with a variable number of peptide-HaloTag fusion proteins, with the primary species containing 3-5 peptide labels (
Binding kinetic studies were performed to establish maximum light output and signal duration of the fully complemented system as show in
Lyophilized, Single-Reagent Tripartite Immunoassays in Vials
To evaluate the potential application of lyophilization for preservation of the entire IL-6 tripartite immunoassay in a single vial, formulations containing peptide labeled antibodies (SmTrip9 Pep521 (SEQ ID NO: 16) and SmTrip10 Pep289 (SEQ ID NO: 17)), LgTrip 3546 (SEQ ID NO: 12), and furimazine were prepared. The 20× stock formulations are as follows:
Formulation A: 20 mM HEPES buffer (pH 8.0), 90 mM glycine, 20 mM histidine, 25 mg/ml sucrose, 0.01% polysorbate 80, 0.6 ug/ml clone 5IL6 antibody labeled with HaloTag-SmTrip9 Pep521 (SEQ ID NO: 16), 1.2 ug/ml 505E A12 A3 antibody labeled with HaloTag-SmTrip10 Pep289 (SEQ ID NO: 17), and 20 μM LgTrip 3546 (SEQ ID NO: 17).
Formulation B: 20 mM HEPES buffer (pH 8.0), 90 mM glycine, 20 mM histidine, 25 mg/ml sucrose, 0.01% polysorbate 80, 0.6 ug/ml clone 5IL6 antibody labeled with HaloTag-SmTrip9 Pep521 (SEQ ID NO: 16), 1.2 ug/ml 505E A12 A3 antibody labeled with HaloTag-SmTrip10 Pep289 (SEQ ID NO: 17), 20 μM LgTrip 3546 (SEQ ID NO: 12), and 100 μM furimazine in ethanol.
Formulation C: 5 mM azothiothymine, 5 mM ascorbic acid, 2.5% pullulan w/v, 20 mM HEPES buffer (pH 8.0), 90 mM glycine, 20 mM histidine, 25 mg/ml sucrose, 0.01% polysorbate 80, 0.6 ug/ml clone 5IL6 antibody labeled with HaloTag-SmTrip9 Pep521 (SEQ ID NO: 16) 1.2 ug/ml 505E A12 A3 antibody labeled with HaloTag-SmTrip10 Pep289 (SEQ ID NO: 17), 20 μM LgTrip 3546 (SEQ ID NO: 12), and 100 μM furimazine in ethanol.
One mL aliquots of 20× stock solution was dispensed into 10 mL amber glass vials, and a runner stopper was partially inserted into the vial. Vials were loaded into the lyophilizer (Virtis Genesis 12EL lyophilizer) with shelves pre-chilled to 4.7° C. Product then underwent a freezing step with a shelf temperature of −50° C. for 2 hr after which time the condenser step started. During the run, the condenser temperature ran between −5° C. and −87° C. A vacuum pulled down ran next at the pressure set-points of 75 and 200 mTorr. Sublimation lasted ˜7.5 hr, and desorption lasted ˜16.1 hr. At the end of the lyophilization process, the vials were back-filled with nitrogen and sealed with fully inserted stoppers at −600 Torr of pressure.
Vials were stored at 25° C. and tested at various timepoints post-lyophilization. For activity-based assays, single-reagent cakes were reconstituted with 10 mL of PBS containing 0.01% BSA. The vials were shaken manually and allowed to equilibrate at room temperature for 5 minutes. 50 μl of the reconstituted substrate was added to 50 μl of recombinant human IL-6 (source) reconstituted in the same BSA buffer. Formulation A requires the addition of furimazine, in which NANOGLO Live Cell Substrate (Promega N205) was used. Assays were performed in solid, white, nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega) collecting total luminescence using kinetic or endpoint reads, depending on the experiment.
To determine the lyophilized assay compatibility with complex human matrices, lyophilized cakes produced with formulation C were reconstituted in PBS (pH 7.0) containing 0.01% BSA. 50 μl was added to wells of 96-well microtiter plates containing 50 ul of rhIL-6 in 20% normal pooled human serum, citrate collected plasma, or urine. In all experiments, plates were incubated at room temperature for 90 minutes. Final concentration of the assay reagents in all experiments were 60 ng/ml SmTrip10-labeled antibody, 30 ng/ml SmTrip9-labeled antibody, 1 μM LgTrip 3546, and 5 μM furimazine. Luminescence was analyzed.
Lyophilized, Single-Reagent Tripartite Immunoassays in Pre-Filled, 96-Well Microtiter Plates
To evaluate the potential application of lyophilization for preservation of the entire IL-6 NanoTrip (tripartite NanoLuc) immunoassay directly into a 96-well microtiter plates, formulations containing 5 mM azothiothymine, 5 mM ascorbic acid, 2.5% pullulan w/v, 20 mM HEPES buffer (pH 8.0), 90 mM glycine, 20 mM histidine, 25 mg/ml sucrose, 0.01% polysorbate 80, 0.12 ug/ml clone 5IL6 antibody labeled with HaloTag-SmTrip9 Pep521 (SEQ ID NO: 16), 0.24 ug/ml 505E A12 A3 antibody labeled with HaloTag-SmTrip10 Pep289 (SEQ ID NO: 17), 4 μM LgTrip 3546 (SEQ ID NO: 12), and 100 μM furimazine in ethanol (same as formulation C in the previous example, but with a 4× reagent addition instead of a 20× stock reagent as used in the vials) were used.
Approximately 25 μl aliquots of 4× stock solution was dispensed into 96-well microtiter plates. Two types of plates were used: non-binding surface (Costar 3600) and non-treated surface (Costar 3912). Plates were loaded into the lyophilizer (Virtis Genesis 12EL lyophilizer) with shelves pre-chilled to 4.7° C. Product then underwent a freezing step with a shelf temperature of −50° C. for 2 hr after when time the condenser step started. During the run, the condenser temperature ran between −5° C. and −87° C. A vacuum pull down ran next at the pressure set-points of 75 and 200 mTorr. Sublimation lasted ˜7.5 hr, and desorption lasted ˜16.1 hr. At the end of the lyophilization process, the plates were back-filled with nitrogen and sealed with adhesive plate cover.
Testing the Effects of Individual Excipients in Formulations Using the Solution-Based, Homogeneous IL-6 Tripartite Immunoassay
To determine the effects of assay performance of individual excipients used in the lyophilized formulations for the single-reagent NanoTrip (tripartite NanoLuc) immunoassays, the IL-6 model system in the solution-based assay was used with the effects of various excipients analyzed.
Creating a Solution-Based and Lyophilized, Single-Reagent Tripartite Immunoassays in Vials for the Target Analyte Human Cardiac Troponin I
The basic principle of the homogeneous NanoTrip (NanoLuc tripartite) cardiac troponin I immunoassay is depicted in
Genetic fusions containing SmTrip9 Pep521 (SEQ ID NO: 16) or SmTrip10 Pep289 (SEQ ID NO: 17) separated by either a 2X or 3X Gly-Ser-Ser-Gly linker to the amino terminus of HaloTag was achieved using the pFN29A HIS6HaloTag T7 Flexi Vector (Promega). Glycerol stocks of E. coli expressing HisTag-HaloTag fusion protein were used to inoculate 50 mL starter cultures, which were grown overnight at 37° C. in LB media containing 25 ug/ml kanamycin. Starter cultures were diluted 1:100 into 500 mL fresh LB media, containing 25 ug/mL kanamycin, 0.12% glucose, and 0.2% rhamnose. Cultures were grown for 22-24 h at 25° C. Cells were pelleted by centrifugation (10,000 rpm) for 30 min at 4° C. and re-suspended in 50 mL PBS. 1 mL protease inhibitor cocktail (Promega), 0.5 mL RQ1 DNase (Promega), and 0.5 mL of 10 mg/mL lysozyme (Sigma) were added, and the cell suspension was incubated on ice with mild agitation for 1 h. Cells were lysed by sonication at 15% power at 5 s intervals for 1.5 min (3 min total) and subsequently centrifuged at 10,000 rpm for 30 min at 4° C. Supernatant was collected, and protein purified using HisTag columns (GE) following the manufacturer's recommended protocol. Protein was eluted using 500 mM imidazole, dialyzed in PBS, characterized using SDS-PAGE gel and was >95% pure. Proteins were stored in 50% glycerol at −20° C.
To chemically conjugate the antibodies to the HaloTag-peptide fusion proteins, antibodies were buffered exchanged 2× into 10 mM sodium bicarbonate buffer (pH 8.5) using Zeba spin desalting columns (ThermoFisher). Antibodies were then primed with 200 μM amine reactive HaloTag Succinimidyl Ester (04) Ligand (Promega) for 2 hr shaking at 1000 rpm at 22° C. Unreacted ligand was removed with two passes through Zeba spin columns in PBS buffer. Then, antibodies were covalently labeled with 30 μM of the HaloTag fusion protein overnight at 4° C. while shaking. Excess unreacted HaloTag fusion protein was removed using HaloLink Resin (Promega). Non-denaturing SDS-PAGE gel was used to characterize the conjugated antibodies. Anti-human cardiac troponin I monoclonal antibodies used in the human cardiac troponin I immunoassay were recombinant rabbit clone 1H11L19 (Invitrogen) and monoclonal mouse antibody clone 16A11 (Invitrogen).
To evaluate the potential application of lyophilization for preservation of the entire cardiac troponin I tripartite immunoassay in a single vial, formulations containing the peptide labeled antibodies (SmTrip9 Pep521 (SEQ ID NO: 16) and SmTrip10 Pep289 (SEQ ID NO: 17)), LgTrip 3546 (SEQ ID NO: 12), and furimazine were prepared. The 20× stock formulations are as follows:
Approximately, 5 mM azothiothymine, 5 mM ascorbic acid, 2.5% pullulan w/v, 20 mM HEPES buffer (pH 8.0), 90 mM glycine, 20 mM histidine, 25 mg/ml sucrose, 0.01% polysorbate 80, 0.08 ug/ml clone 1H11L19 antibody labeled with HaloTag-SmTrip9 Pep521 (SEQ ID NO: 16), 1.6 ug/ml of clone 16A11 antibody labeled with HaloTag-SmTrip10 Pep289 (SEQ ID NO: 17), 20 μM LgTrip 3546 (SEQ ID NO: 12), and 200 μM furimazine (Promega NANOGLO substrate N113).
One mL aliquots of 20× stock solution were dispensed into 10 mL amber glass vials, and a runner stopper was partially inserted into the vial. Vials were loaded into the lyophilizer (Virtis Genesis 12EL lyophilizer) with shelves pre-chilled to 4.7° C. Product then underwent a freezing step with a shelf temperature of −50° C. for 2 hr after which time the condenser step started. During the run, the condenser temperature ran between −5° C. and −87° C. A vacuum pull down ran next at the pressure set-points of 75 and 200 mTorr. Sublimation lasted ˜7.5 hr, and desorption lasted ˜16.1 hr. At the end of the lyophilization process, the vials were back-filled with nitrogen and sealed with fully inserted stoppers at −600 Torr of pressure.
For activity-based assays, single-reagent cakes were reconstituted with 10 mL of PBS containing 0.01% BSA. The vials were shaken manually and allowed to equilibrate at room temperature for 5 minutes. 50 μl of the reconstituted single-reagent cardiac troponin I NanoTrip (tripartite NanoLuc) immunoassay was added to 50 μl of recombinant human cardiac troponin I (Fitzgerald) that was reconstituted in the same BSA buffer or with 20% human serum diluted in General Serum Diluent (Immunochemistry Technologies). Assays were performed in solid, white, nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega) collecting total luminescence using an endpoint read.
Investigating and Mitigating the Effects of Complex Sample Matrices on Tripartite Immunoassay Performance
A solution-based, homogeneous IL-6 NanoTrip (tripartite NanoLuc) immunoassay was tested to determine if the assay was compatible with human sample types commonly analyzed for clinical biomarkers, and factors in the samples that might affect the performance of the assay and possible solutions to mitigate these effects were investigated. This is critical because sample matrix interference effects in immunoassays, defined as the effect of a substance present in the sample that alters the correct value of the result, are a common phenomenon especially in homogenous formats due to the removal of the wash steps.
Reagents used for the following experiments were the HaloTag-peptide labeled antibodies described in Example 19. 30 ng/ml clone 5IL6 antibody labeled with HaloTag-SmTrip9 Pep521 (SEQ ID NO: 16), 60 ng/ml 505E A12 A3 antibody labeled with HaloTag-SmTrip10 Pep289 (SEQ ID NO: 17), 1 μM LgTrip 3546 (SEQ ID NO: 12), and NANOGLO Live Cell Substrate (Promega N205) or NANOGLO substrate (Promega N113), which were used according to the manufacturer's instructions, but were diluted in the given buffer for that experiment. Assays were performed+/−50 ng/ml recombinant human IL-6 (R&D Systems) with assay backgrounds, and Bmax analyzed. Assays were allowed to incubate on the bench for 90 minutes prior to addition of substrate. Assays were performed in solid, white, nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega) collecting total luminescence using an endpoint read.
Next, the effects of endogenous IgG in human serum samples had on assay performance was determined. Using the solution-based, homogeneous IL-6 NanoTrip assay+/−50 ng/ml rhIL-6 in General Serum Diluent, the bioluminescent response when running the assay in normal, pooled human serum or in serum that had been depleted of endogenous IgG was analyzed.
Next, the effects of blood biochemistry on the solution-based, homogenous IL-6 tripartite immunoassay was investigated using the VeriChem reference plus chemistry kit, which contains the following:
The IL-6 NanoTrip assay was run in the presence of Level A-E diluted in general serum diluent and using NANOGLO Live Cell Substrate (Promega N205) to determine the effects of increasing these blood chemistry components on assay performance.
To determine the effects of urine on the solution-based, homogeneous IL-6 NanoTrip immunoassay performance, a IL-6 screening assay in the presence of increasing normal, pooled human urine diluted in General Serum Diluent and NANOGLO substrate (Promega N113) or NANOGLO Live Cell Substrate (Promega N205) was performed.
Creating a Stable, Lyophilized Substrate and LgTrip Cake Reagent in a Single Vial
To evaluate the potential application of lyophilization for preservation of furimazine, LgTrip and furimazine were paired with LgTrip 3546 used as a general detection reagent for tripartite applications and supplied in a single vial. Formulations containing furimazine, LgTrip 3546 (SEQ ID NO: 12), and furimazine with LgTrip 3546 were prepared. The 20× stock formulations are as follows:
Furimazine only formulation: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% pullulan w/v, 200 μM furimazine in ethanol, and ddH20 millipore
LgTrip 3546 only formulation: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% pullulan w/v, 20 μM LgTrip 3546 (SEQ ID NO: 12), and ddH20 (Millipore)
Furimazine with LgTrip 3546 formulation: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% pullulan w/v, 200 μM furimazine in ethanol, 20 μM LgTrip 3546 (SEQ ID NO: 12) and ddH20 (Millipore).
One mL aliquots of 20× stock solution was dispensed into 10 mL amber glass vials, and a runner stopper was partially inserted into the vial. Vials were loaded into the lyophilizer (Virtis Genesis 12EL lyophilizer) with shelves pre-chilled to 4.7° C. Product then underwent a freezing step with a shelf temperature of −50° C. for 2 hr after when time the condenser step started. During the run, the condenser temperature ran between −5° C. and −87° C. A vacuum pull down ran next at the pressure set-points of 75 and 200 mTorr. Sublimation lasted ˜7.5 hr and desorption lasted ˜16.1 hr. At the end of the lyophilization process, the vials were back-filled with nitrogen and sealed with fully inserted stoppers at −600 Torr of pressure.
Vials were stored at 25° C. or 60° C. and tested at various time points post-lyophilization. For activity-based assays, lyophilized cakes were reconstituted with 10 mL of PBS containing 0.01% BSA. The vials were shaken manually and allowed to equilibrate at room temperature for 5 minutes. 50 μl of the reconstituted substrate was added to 50 μl of purified NANOLUC enzyme (Promega) or dipeptide (SEQ ID NO: 14) that was reconstituted in the same BSA buffer. LgTrip 3546 only formulations required the addition of furimazine in which NANOGLO Live Cell Substrate (Promega N205) was used. Assays were performed in solid, white, nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega) collecting total luminescence using an endpoint read.
Creating a Solution-Based and Lyophilized, Single-Reagent Tripartite Immunoassays in Vials for the Target Analytes Anti-TNFα Biologics
The basic principle of the homogeneous anti-TNFα biologics NanoTrip (tripartite NanoLuc) immunoassay is depicted in
6×His-TNFα-J5GS-HiBiT (ATG-3998). Genetic fusions containing the SmTrip10 (SEQ ID NO: 15) separated by a 15GS linker (SSSGGGGSGGGSSGG) to the carboxyl-terminus of TNFα was achieved using the pF4Ag CMV Flexi Vector (Promega). Purified plasmid DNA of the TNFα-strand 10 fusion was transformed into Shuffle T7 E. coli K12 (New England Biolabs) and plated at a 1:100 dilution on an LB plate containing 100 μg/ml ampicillin and incubated overnight at 37° C. A colony from this plate was used to inoculate 50 mL starter cultures, which were grown overnight at 37° C. in LB media containing 100 μg/ml ampicillin. Starter cultures were diluted 1:100 into 500 mL fresh LB media containing 100 μg/ml ampicillin and were incubated at 37° C. until it reached an OD of 0.6, at which time a final concentration of 1 mM IPTG was added to the sample. After IPTG inoculation, cultures were grown overnight at 25° C. Cells were pelleted by centrifugation (10,000 rpm) for 30 min at 4° C. and re-suspended in 50 mL TBS, 1 mL protease inhibitor cocktail (Promega), 0.5 mL RQ1 DNase (Promega), and 1 mL of 10 mg/mL lysozyme (Sigma), and the cell suspension was incubated on ice with mild agitation for 1 h. Cells were lysed by three freeze-thaw cycles from −80° C. freezer to a 37° C. water bath and subsequently centrifuged at 10,000 rpm for 30 min at 4° C. Supernatant was collected and protein was purified using Ni Sepharose 6 Fast Flow resin (GE), following manufacturer's recommended protocol. Protein was eluted using a step-wise imidazole elution starting at 100 mM imidazole and reaching up to 500 mM imidazole, dialyzed in TBS, characterized using SDS-PAGE gel and was >95% pure. Proteins were stored in 50% glycerol at −20° C.
SmTrip9(521)-15GS-PtnG-6×His (ATG4002). Genetic fusions containing the SmTrip9 (SEQ ID NO: 13) separated by a linker (GSSGGGGSGGGGSSG) to the amino terminus of Protein G was achieved using the pF1A T7 Flexi Vector (Promega). Glycerol stocks of E. coli expressing SmTrip9(521)-PtnG fusion protein was used to inoculate 50 mL starter cultures, which were grown overnight at 37° C. in LB media containing 100 μg/ml ampicillin. Starter cultures were diluted 1:100 into 500 mL fresh LB media, containing 100 μg/mL ampicillin, 0.15% glucose, and 0.1% rhamnose. Cultures were grown for 16-24 h at 25° C. Cells were pelleted by centrifugation (10,000 rpm) for 30 min at 4° C. and re-suspended in 50 mL TBS. 1 mL protease inhibitor cocktail (Promega), 0.5 mL RQ1 DNase (Promega), and 1 mL of 10 mg/mL lysozyme (Sigma) were added, and the cell suspension was incubated on ice with mild agitation for 1 h. Cells were lysed by three freeze-thaw cycles from −80° C. freezer to a 37° C. water bath and subsequently centrifuged at 10,000 rpm for 30 min at 4° C. Supernatant was collected and protein purified using HisTag columns (GE), following manufacturer's recommended protocol. Protein was eluted using gradient elution with a 500 mM imidazole final concentration, dialyzed in TBS, characterized using SDS-PAGE gel and was >95% pure. Proteins were stored in 50% glycerol at −20° C.
To evaluate the potential application of lyophilization for preservation of the entire anti-TNFαTNFα biologics, NanoTrip and NanoBiT immunoassays in single vial formulations containing peptide-labeled fusion proteins and LgTrip 3546 (SEQ ID NO: 12; for NanoTrip assays) and furimazine were prepared. The 20× stock formulations are as follows:
NanoTrip anti-TNFα biologics immunoassay: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% w/v pullulan, ddH20 (Millipore), 200 μM furimazine in ethanol, 20 μM LgTrip 3546 protein (SEQ ID NO:12), 200 nM protein G-SmTrip9 Pep521 (SEQ ID NO: 16) fusion protein, and 200 nM TNFα-SmTrip10 Pep289 (SEQ ID NO:17) fusion protein.
NanoBiT anti-TNFα biologics immunoassay: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% w/v pullulan, ddH20 (Millipore), 200 μM furimazine in ethanol, 200 nM protein G-SmBiT (SEQ ID NO:10) fusion protein, and 200 nM TNFα-LgBiT (SEQ ID NO: 12) fusion protein.
One mL aliquots of 20× stock solution was dispensed into 10 mL amber glass vials, and a runner stopper was partially inserted into the vial. Vials were loaded into the lyophilizer (Virtis Genesis 12EL lyophilizer) with shelves pre-chilled to 4.7° C. Product then underwent a freezing step with a shelf temperature of −50° C. for 2 hr after which time the condenser step started. During the run, the condenser temperature ran between −5° C. and −87° C. A vacuum pull down ran next at the pressure set-points of 75 and 200 mTorr. Sublimation lasted ˜7.5 hr and desorption lasted ˜16.1 hr. At the end of the lyophilization process, the vials were back-filled with nitrogen and sealed with fully inserted stoppers at ˜600 Torr of pressure.
For activity-based assays, single-reagent cakes were reconstituted with 10 mL of PBS containing 0.01% BSA. The vials were shaken manually and allowed to equilibrate at room temperature for 5 minutes. 50 μl of the reconstituted single-reagent anti-TNFα biologics NanoTrip and NanoBiT immunoassays were added to 50 μl of Remicade in a titration that was reconstituted in the same BSA buffer. Assays were performed in solid, white, nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega) collecting total luminescence using a kinetic read.
Testing the thermal stability of these lyophilized, single-reagent anti-TNFα biologics NanoTrip and NanoBiT immunoassays when stored at ambient temperatures indicated that both assays, when reconstituted in 0.01% BSA in PBS (pH 7.0) in the presence or absence of 100 nM Remicade, displayed shelf stability and a significant increase in signal when the analyte Remicade is present. Results are shown in
Developing Stable, Lyophilized Tripartite and NanoBiT Immunoassay Using a Split-Reagent Approach
To evaluate the potential application of lyophilization for preservation of separate components of the anti-TNFα biologics, NanoTrip and NanoBiT immunoassays that are then combined in a single vial formulations containing the peptide labeled fusion proteins and LgTrip 3546 (SEQ ID NO: 12; for NanoTrip assays) and furimazine were prepared. The 20× stock formulations are as follows:
NanoBiT anti-TNFα biologics immunoassay:
Furimazine with LgBiT-TNFα: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% w/v pullulan, ddH20 (Millipore), 200 μMfurimazine in ethanol, and 200 nM TNFα-LgBiT (SEQ ID NO: 12) fusion protein.
NanoBiT protein G: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% w/v pullulan, ddH20 millipore, 200 nM protein G-SmBiT (SEQ ID NO: 10) fusion protein
NanoTrip Anti-TNFα Biologics Immunoassay:
Furimazine with LgTrip 3546: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% w/v pullulan, ddH20 (Millipore), 200 μMfurimazine in ethanol, 20 μM LgTrip 3546 protein (SEQ ID NO: 12),
Protein G with TNFα: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% w/v pullulan, ddH20 (Millipore), 200 nM protein G-SmTrip9 Pep521 (SEQ ID NO: 16) fusion protein, and 200 nM TNFα-SmTrip10 Pep289 (SEQ ID NO: 17) fusion protein.
Formulations were lyophilized as separate components then manually combined to create the complete immunoassay. Cakes were reconstituted with Opti-MEM (Gibco), and 50 ul added to 50 μl of Remicade in a dose titration. Assays were performed in solid, white, nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega) collecting total luminescence using a kinetic read.
Developing a Cell-Based, Homogeneous Tripartite Assay for the Quantitation of Anti-EGFR Biologics
A bulk transfection was performed on HEK293 cells by preparing a 10 μg/ml solution of DNA with a 1:10 dilution of IL6-VSHiBiT-15GS-EGFR (GSSGGGGSGGGGSS) (ATG-4288) and pGEM3Z carrier DNA (Promega). FuGENE HD was added to the DNA mixture to form a lipid:DNA complex. This complex was added to HEK293 cells with an adjusted cell density of 2×105 cells/ml and incubated at 37° C. and 5% CO2 overnight.
Transfected HEK293 cells were added to 96-well NBS plates (a separate plate for each SmTrip-15GS-G being tested) at a final concentration of 2×105 cells/well. A reagent mixture of LgTrip 3546 and SmTrip9-G was added to the cells at a final concentration of 1 μM LgTrip 3546 and 10 nM SmTrip9-15GS-G. A 24-point panitumumab titration was added to each well with a final starting concentration of 100 nM and diluted 1:2 with a final ending concentration of 0 nM. All plates were covered and incubated for an hour at 37° C. and 5% CO2. NANOLUC Live Cell Substrate was added to all wells at a final concentration of 10 and luminescence of each plate was subsequently read on a luminometer. The following SmTrip9-G constructs were tested: ATG4002 SmTrip9(521)-15GS-G (SEQ ID NO: 724); ATG4496 SmTrip9(743)-15GS-G (SEQ ID NO: (726); ATG4558 SmTrip9(759)-15GS-G (SEQ ID NO: 728); and ATG4551 SmTrip9(760)-15GS-G (SEQ ID NO: 730). Each configuration was successful in quantitatively detecting panitumumab.
Testing Various SmTrip9-Protein G Fusion Proteins in Solution-Based, Homogeneous Anti-TNFα Biologics Tripartite Immunoassays
To evaluate the potential application of lyophilization for preservation of the entire anti-TNFα biologics, NanoTrip immunoassays in single vial formulations containing peptide-labeled fusion proteins and LgTrip 3546 (SEQ ID NO: 12) and furimazine were prepared. The 20× stock formulations are as follows:
NanoTrip anti-TNFα biologics immunoassay: 5 mM azothiothymine, 5 mM ascorbic acid, 2.75% w/v pullulan, ddH20 (Millipore), 200 μM furimazine in ethanol, 20 μM LgTrip 3546 protein (SEQ ID NO:12), 200 nM protein G-SmTrip9 variant fusion protein, and 200 nM TNFα-SmTrip10 Pep289 (SEQ ID NO:17) fusion protein.
One mL aliquots of 20× stock solution was dispensed into 10 mL amber glass vials, and a runner stopper was partially inserted into the vial. Vials were loaded into the lyophilizer (Virtis Genesis 12EL lyophilizer) with shelves pre-chilled to 4.7° C. Product then underwent a freezing step with a shelf temperature of −50° C. for 2 hr after which time the condenser step started. During the run, the condenser temperature ran between −5° C. and −87° C. A vacuum pull down ran next at the pressure set-points of 75 and 200 mTorr. Sublimation lasted ˜7.5 hr and desorption lasted ˜16.1 hr. At the end of the lyophilization process, the vials were back-filled with nitrogen and sealed with fully inserted stoppers at −600 Torr of pressure.
Direct-Labeling of Antibodies Via Reactive Peptides for Development of Solution-Based, Homogenous IL-6 Immunoassays
The basic principle of homogeneous NanoLuc tripartite immunoassays with directly-labeled antibodies is depicted in
SmTrip9 variants such as Pep693 (SEQ ID NO: 20), Pep895 (SEQ ID NO: 24), and Pep929 (SEQ ID NO: 25) or SmTrip10 variants such as Pep691 (SEQ ID NO: 18) and Pep692 (SEQ ID NO: 19) were individually dissolved in DMF to 5 mM. Antibodies were buffered exchanged 2× into 10 mM sodium bicarbonate buffer (pH 8.5) using Zeba spin desalting columns (ThermoFisher). Subsequently, these antibodies were combined with 20× molar excess of a reactive peptide for 1 hr at 4° C. while shaking in order to covalently label the proteins. Unreacted label was removed with two passes through Zeba spin columns in PBS buffer. To create the reagents for the exemplary human IL-6 immunoassay, the mouse anti-human IL-6 monoclonal antibodies clone 5IL6 (Thermo cat #M620) and clone 505E 9A12 A3 (Thermo cat #AHC0662) were used. SmTrip9 reactive peptides were used to label antibody 5IL6 while SmTrip10 reactive peptides were used to label antibody 505E. The denaturing SDS-PAGE gel shown in
Components for homogeneous tripartite NanoLuc immunoassays can also be constructed by direct-labeling antibodies with SmTrip9 or SmTrip10 variants that contain a fluorophore such as tetramethylrhodamine (TMR). This is depicted schematically in
SulfoSE-PEG3-SmTrip9 Pep693 (HW-0984)
PEG3 bis Sulfo-SE
3,3′-((oxybis(ethane-2,1-diyl))bis(oxy))dipropionic acid (55 mg, 0.22 mmol) was dissolved in anhydrous DMF, and then diisopropylethylamine (120 mg, 0.88 mmol) and HATU (176 mg, 0.45 mmol) added. The mixture was stirred for five minutes. Meanwhile, N-hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid (90 mg, 0.46 mmol) was dissolved in 5 ml DMSO and then added to the previous solution dropwise. The mixture was stirred for another hour until LC-MS shows disappearance of acid. The solution was directly used in the next step. Calculated: m/z=603.05 [M−]; measured (ESI): m/z=603.04 [M−].
SulfoSE-PEG3-SmTrip9 Pep693 (HW-0984)
SmTrip9 Pep693 (GRMLFRVTINSWR, 27 mg, 0.045 mmol) was dissolved in DMF. The solution was then added to the previous PEG3 bis Sulfo-SE solution. The mixture was then stirred for another hour and directly purified by preparative HPLC. Calculated: m/z=1022.98 [M+2H]2+; measured (ESI): m/z=1023.09 [M+2H]2+.
SulfoSE-PEG3-SmTrip10 Pep691 (HW-0977)
HW-0977 was synthesized by the same method as HW-0984. Calculated: m/z=892.93 [M+2H]2+; measured (ESI): m/z=893.61 [M+2H]2+.
SulfoSE-PEG3-SmTrip9 Pep895 (HW-1010)
HW-1010 was synthesized by the same method as HW-0984. Calculated: m/z=1016.51 [M+2H]2+; measured (ESI): m/z=1016.92 [M+2H]2+.
SulfoSE-PEG3-SmTrip9 Pep929 (HW-1055)
HW-1055 was synthesized by the same method as HW-0984. Calculated: m/z=1114.06 [M+2H]2+; measured (ESI): m/z=1113.95 [M+2H]2+.
SulfoSE-PEG6-SmTrip9 Pep693 (HW-1042)
PEG6 bis Sulfo-SE
Bis PEG6-acid (39 mg, 0.10 mmol) was dissolved in anhydrous DMF and then diisopropylethylamine (53 mg, 0.4 mmol) and HATU (78 mg, 0.20 mmol) added. The mixture was stirred for five minutes. Meanwhile, N-hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid (40 mg, 0.20 mmol) was dissolved in 5 ml DMSO and then added to the previous solution dropwise. The mixture was stirred for another hour until LC-MS shows disappearance of acid. The solution was directly used in the next step. Calculated: m/z=735.13 [M−]; measured (ESI): m/z=735.04 [M].
SulfoSE-PEG6-SmTrip9 Pen693 (HW-1042)
SmTrip9 Pep693 (GRMLFRVTINSWR, 20 mg, 0.013 mmol) was dissolved in DMF. The solution was then added to the previous PEG6 bis Sulfo-SE solution. The mixture was then stirred for another hour and directly purified by preparative HPLC. Calculated: m/z=1089.02 [M+2H]2+; measured (ESI): m/z=1088.94 [M+2H]2+.
SulfoSE-PEG6-SmTrip9 Pep929 (HW-1052)
HW-1052 was synthesized by the same method as HW-1042. Calculated: m/z=1180.10 [M+2H]2+; measured (ESI): m/z=1179.82 [M+2H]2+.
SulfoSE-PEG6-SmTrip10 Pep692 (HW-1053)
HW-1053 was synthesized by the same method as HW-1042. Calculated: m/z=1052.03 [M+2H]2+; measured (ESI): m/z=1051.92 [M+2H]2+.
SulfoSE-PEG6-SmTrip9 Pep895 (HW-1043)
HW-1043 was synthesized by the same method as HW-1042. Calculated: m/z=1082.55 [M+2H]2+; measured (ESI): m/z=1082.34 [M+2H]2+.
SulfoSE-PEG3-SmTrip9 Pep938-TAMRA (HW-0992)
TAMRA-Maleimide
5-TAN/IRA (50 mg, 0.116 mmol) was dissolved in DMF. Diisopropylethylamine (45 mg, 0.128 mmol) was added followed by TSTU (38 mg, 0.128 mmol). The mixture was stirred for 20 min, 1-(2-aminoethyl)-1H-pyrrole-2,5-dione (18 mg, 0.128 mmol) added, and the resulting reaction mixture was stirred for another hour and directly purified by preparative HPLC. Calculated: m/z=553.20 [M+H]+; measured (ESI): m/z=553.40 [M+H]+.
SmTrip9 Pep938-TAMRA
TAMRA-Maleimide (8 mg, 0.014 mmol) was dissolved in DMF. A solution of SmTrip9 (Pep938) (GRMLFRVTINSWRC, 25 mg, 0.014 mmol) in PBS buffer (pH 7.4, 200 mM) was added. The reaction mixture was stirred for two hours and directly purified by preparative HPLC. Calculated: m/z=1146.05 [M+2H]2+; measured (ESI): m/z=1146.33 [M+2H]2+.
SulfoSE-PEG3-SmTrip9 Pep938-TAMRA (HW-0992)
SmTrip9 Pep938-TAMRA (8.5 mg, 0.0038 mmol) was dissolved in DMF. The solution was then added to PEG3 bis Sulfo-SE prepared as shown in synthesis of HW-0984. The reaction mixture was stirred for two hours and directly purified by preparative HPLC. Calculated: m/z=901.05 [M+3H]3+; measured (ESI): m/z=901.20 [M+3H]3+.
SulfoSE-PEG3-Strnd 9 (Pep937)-TAMRA (HW-0987)
HW-0987 was synthesized by the same method as HW-0992. Calculated: m/z=814.03 [M+3H]3+; measured (ESI): m/z=814.40 [M+3H]3+.
SulfoSE-PEG3-SmTrip9 Pep938-SA (HW-1050)
SmTrip9 Pep938-SA
SmTrip9 Pep938 (GRMLFRVTINSWR, 26 mg, 0.015 mmol) was dissolved in DMSO. 1-(3-Sulfopropyl)-2-vinylpyridinium Hydroxide Inner Salt (3.40 mg 0.015 mmol) was dissolved in phosphate buffer (pH=7.4, 100 mM) and was added slowly to the peptide solution. The mixture was stirred for another three hours and directly purified by preparative HPLC. Calculated: m/z=983.48 [M+2H]2+; measured (ESI): m/z=983.39 [M+2H]2+.
SulfoSE-PEG3-SmTrip9 Pep938-SA (HW-1050)
SmTrip9 Pep938-SA (10 mg, 0.005 mmol) was dissolved in DMF. The solution was then added to PEG6 bis Sulfo-SE prepared as shown in HW-0984. The reaction mixture was stirred for two hours and directly purified by preparative HPLC. Calculated: m/z=1254.05 [M+2H]2+; measured (ESI): m/z=1253.98 [M+2H]2+.
Shown below is a representative scheme for the synthesis of PEG-linked peptide SulfoSE.
Shown below is a representative scheme for the synthesis of PEG-linked peptide SulfoSE linked to a fluorophore.
Investigating Luminescence in Complex Sample Matrices on Performance of Coelenterazine Derivatives JRW-1404 and JRW-1482
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.
The following polypeptide sequences each comprise an N-terminal methionine residue or corresponding ATG codon; polypeptide sequences lacking the N-terminal methionine residue or corresponding ATG codon are also within the scope herein and are incorporated herein by reference.
The following peptide sequences each lack an N-terminal methionine residue; peptide sequences comprising an N-terminal methionine residue are also within the scope herein and are incorporated herein by reference.
LNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRVTINSVSG
WRLFKKIS
LNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRVTINSVTG
YRLFEEIL
LNYFGRPYEGIAVFDG
LNYFGRPYEGIAVFDGKKITTTGTL
LNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD
GVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRV
WNGNKIIDERLITPDGSMLFRVTINSVSGWRLFKKIS
GVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRV
WNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL
GVTPNKLNYFGRPYEGIAVFDG
WNGNKIIDERLITPD
The present application claims priority to and the benefit of U.S. Provisional Patent Application No. 62/832,052, filed Apr. 10, 2019, which is incorporated herein by reference in its entirety and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62832052 | Apr 2019 | US |
