Patent Application
20230408498
Molecular switches that change their conformation upon target analyte binding offer powerful capabilities for biotechnology and synthetic biology.
In one aspect, the disclosure features an antibody construct comprising: an antibody or a binding fragment thereof comprising a first label, wherein the antibody or the binding fragment thereof is linked to a blocking analyte and a second label via a linker, wherein: in the absence of a target analyte, the blocking analyte binds to the antibody or the binding fragment thereof, and in the presence of the target analyte, the target analyte competes with the blocking analyte for binding to the antibody or the binding fragment thereof, and the first and second labels interact to generate a detectable readout that changes depending on whether the blocking analyte is bound by the antibody or the binding fragment thereof, or is not bound by the antibody or the binding fragment thereof.
In some embodiments, the second label is conjugated to the linker. In some embodiments, the second label is conjugated to the blocking analyte. In some embodiments, the first label is a fluorophore and the second label is a quencher. In some embodiments, the first label is a quencher and the second label is a fluorophore. In some embodiments, the first label is a donor fluorophore and the second label is an acceptor fluorophore. In some embodiments, the first label is an acceptor fluorophore and the second label is a donor fluorophore.
In some embodiments, the detectable readout is an optical signal, an electrical signal, an electrochemical signal, a nuclear magnetic resonance signal, or a biological signal. In some embodiments, the optical signal is a fluorescent signal.
In some embodiments, binding of the target analyte to the antibody or the binding fragment thereof increases or decreases the detectable readout.
Also provided is an antibody construct comprising: an antibody or a binding fragment thereof comprising a redox reporter, wherein the antibody or the binding fragment thereof is linked to a blocking analyte and a sensing electrode; wherein: in the absence of a target analyte, the blocking analyte binds to the antibody or the binding fragment thereof, and in the presence of the target analyte, the target analyte competes with the blocking analyte for binding to the antibody or the binding fragment thereof, and the redox reporter and the sensing electrode interact to generate an electrical signal that changes depending on whether the blocking analyte is bound by the antibody or the binding fragment thereof, or is not bound by the antibody or the binding fragment thereof.
In some embodiments, the antibody or the binding fragment thereof is linked to the blocking analyte and the sensing electrode via a linker. In some embodiments, the antibody or the binding fragment thereof is linked to the blocking analyte by a first linker and the antibody or the binding fragment thereof is linked to the sensing electrode via a second linker. In some embodiments, the antibody or the binding fragment thereof is linked to the sensing electrode via a gold-thiol bond. In some embodiments, the sensing electrode is a gold electrode.
In some embodiments, the linker is a double-stranded polynucleotide. In some embodiments, the linker is a single-stranded polynucleotide. In some embodiments, the linker is a partially double-stranded polynucleotide comprising at least one unpaired nucleotide. In some embodiments, the partially double-stranded polynucleotide comprises between 1 and 30 unpaired nucleotides. In some embodiments, the linker is a partially double-stranded polynucleotide, and wherein at least 1% of the length of the linker comprises unpaired nucleotide(s). In some embodiments, between 1% and 50% of the length of the linker comprises unpaired nucleotide(s).
In some embodiments, the unpaired nucleotides are located at the 5′ terminus or the 3′ terminus of the linker.
In some embodiments, the length of the linker is at least 10 nucleotides. In some embodiments, the length of the linker is between 10 and 100 nucleotides.
In some embodiments, the linker comprises one or more mismatched nucleotides.
In some embodiments, the antibody is covalently conjugated to the linker via an Fc region of the antibody.
In some embodiments, the blocking analyte and the target analyte are the same.
In some embodiments, blocking analyte is a structural analog of the target analyte.
In some embodiments, a binding affinity of the antibody to the target analyte is between 90% and 110% of a binding affinity of the antibody to the blocking analyte. In some embodiments, the antibody has a higher binding affinity for the target analyte than for the blocking analyte. In some embodiments, the antibody has a lower binding affinity for the target analyte than for the blocking analyte.
In some of the embodiments described herein, the blocking analyte can bind to the antibody in the antibody construct independent of the antibody's cognate antigen type. In particular embodiments, the blocking analyte is Protein M, Protein A, Protein G, or Protein L (e.g., Protein M).
Also provided is an antibody construct comprising: (a) two detecting strands, wherein a first detecting strand comprises a first antibody or a binding fragment thereof, and a second detecting strand comprises a second antibody or a binding fragment thereof; and (b) a first label and a second label, wherein the detecting strands or portions thereof are complementary and hybridize to each other or one or more scaffold strands, and wherein in the presence of the target analyte, the first antibody and the second antibody bind to two different epitopes on the target analyte, and the first and second labels interact with each other to generate a detectable readout compared to when there is an absence of the target analyte.
In some embodiments, the two detecting strands hybridize to a scaffold strand, thereby linking the two detecting strands. In some embodiments, the two detecting strands hybridize to a single scaffold strand. In some embodiments, two detecting strands hybridize to separate scaffold strands which scaffold strands hybridize to each other.
In some embodiments, the two detecting strands hybridize to each other, thereby linking the two detecting strands.
In some embodiments, the first label is linked to the first antibody and the second label is linked to the second antibody. In some embodiments, the first label and/or second label is linked to a scaffold strand. In some embodiments, the first label is linked to a first label oligonucleotide that is hybridized to a scaffold strand and second label is linked to a second label oligonucleotide that is hybridized to a scaffold strand. In some embodiments, the first label is linked to a first scaffold strand and the second label is linked to a second scaffold strand. In some embodiments, in the presence of the target analyte, a portion of the first scaffold strand and a portion of the second scaffold strand hybridize to each other.
In some embodiments, the antibody construct comprises two scaffold strands, wherein a first scaffold strand and a second scaffold strand are complementary and hybridize to each other. In some embodiments, the first detecting strand or a portion thereof, the second detecting strand or a portion thereof, and the second scaffold strand or a portion thereof are complementary and hybridize to the first scaffold strand in a linear order with the second scaffold strand between the first and second detecting strands. In some embodiments, the first scaffold strand is at least 100 nucleotides long In some embodiments, the first scaffold strand is between 100 and 200 nucleotides long.
In some embodiments, the second scaffold strand is at least 20 nucleotides long. In some embodiments, the second scaffold strand is between 20 and 180 nucleotides long. In some embodiments, each of the first and second detecting strands is at least 20 nucleotides long. In some embodiments, each of the first and second detecting strands is between 20 and 40 nucleotides long.
In some embodiments, the antibody construct comprises three or more scaffold strands. In some embodiments, each of the three or more scaffold strands is independently at least 100 nucleotides long. In some embodiments, each of the three or more scaffold strands is independent between 100 and 200 nucleotides long. In some embodiments, pairs of the scaffold strands or portions thereof are complementary and hybridize to each other.
In some embodiments, the first antibody is covalently conjugated to the first detecting strand via an Fc region of the first antibody, and wherein the second antibody is covalently conjugated to the second detecting strand via an Fc region of the second antibody.
In some embodiments, the scaffold strands comprise one or more mismatched nucleotides.
In some embodiments, the detectable readout is an optical signal, an electrical signal, an electrochemical signal, a nuclear magnetic resonance signal, or a biological signal. In some embodiments, the optical signal is a fluorescent signal.
In some embodiments, the binding of the target analyte to the first and second antibodies increases or decreases the detectable readout.
Also provided is a method of detecting a target analyte in a sample, comprising: (1) contacting the sample with an antibody construct as described above or elsewhere herein; and (2) measuring binding of the antibody construct to the target analyte using a detectable readout from the antibody construct.
In some embodiments, the detectable readout is an optical signal, an electrical signal, an electrochemical signal, a nuclear magnetic resonance signal, or a biological signal. In some embodiments, the optical signal is a fluorescent signal.
In some embodiments, the sample is a biological sample.
Also provided is a method of adjusting kinetics and/or effective binding affinity of an antibody construct, comprising: (1) generating an antibody construct as described above or elsewhere herein; (2) measuring binding of the antibody construct to a target analyte; (3) changing one or more components of the antibody construct; (4) re measure binding of the antibody construct to the target analyte; and(5) optionally repeat steps (3) and (4) until the desired kinetics and/or effective binding affinity of the antibody construct is reached.
In some embodiments, step (3) comprises increasing or decreasing the length of the linker. In some embodiments, step (3) comprises introducing one or more mismatched nucleotides into the linker. In some embodiments, step (3) comprises changing the identity of the blocking analyte. In some embodiments, step (3) comprises increasing or decreasing the length of one or more scaffold strands. In some embodiments, step (3) comprises introducing one or more mismatched nucleotides into one or more scaffold strands. In some embodiments, step (3) comprises introducing a structural motif into the linker.
In some embodiments, the structural motif is a DNA/RNA duplex, a polynucleotide hairpin, a G-quadruplex, or an i-motif.
The disclosure provides antibody constructs that can be used as programmable affinity reagents for applications in, for example, molecular diagnostics, biomedical imaging, point-of-care devices, and home-use tests. Depending on the assay format, the antibody construct can enable reversible, rapid, quantitative, sensitive, and multiplexed biomolecule detection in complex biological samples.
The antibody constructs described herein can bind specifically to biological or chemical target analytes (e.g., small molecules, peptides, drugs, glycans, proteins, and DNA, etc.). Upon target analyte binding, the antibody construct changes its conformational state to produce a detectable readout. As described herein, many configurations of the antibody constructs are possible. For example, an antibody construct can be a single antibody construct or a double antibody construct. An antibody construct can have two conformational states, i.e. a bi-state molecular switch. The population distribution between the two states, as well as the transition kinetics between the states, can be programmed by designing and tailoring various components in the antibody constructs described herein, such as the linker, the scaffold strands, and the detecting strands. The conformation of the antibody construct can be dynamically modulated by environmental factors such as the presence of the target analytes. The changes in population distribution between the conformational states can be transduced to detectable readouts.
In some embodiments of a single antibody construct, the antibody construct can contain an antibody and a blocking analyte. The presence of target analyte can disrupt the interaction between the antibody and blocking analyte and switch the construct to the “ON” state. In some embodiments of the double antibody construct, the antibody construct can contain two antibodies targeting different epitopes on the same target analyte. The present of target analyte can induce a sandwich complex formation and change the construct conformation.
As used herein, the term “antibody construct” refers to a molecular structure that changes its conformation upon binding to a target analyte. In one example, an antibody construct can contain an antibody comprising a first label, in which the antibody is linked to a blocking analyte and a second label by way of a linker. In another example, an antibody construct can contain at least two scaffold strands, two detecting strands, and two labels.
As used herein, the term “linker” refers to a linkage between two elements, e.g., an antibody and a blocking analyte in an antibody construct. A linker can be a polymer, e.g., a polynucleotide or a polypeptide, that can provide space and/or flexibility between an antibody and a blocking analyte in the antibody construct.
As used herein, the term “label” refers to an agent that can produce a measurable signal or block a measurable signal. For example, a label can be a fluorophore or a quencher. In some embodiments, when two labels interact to form a Fluorescence Resonance Energy Transfer (FRET) pair, a label can be a donor fluorophore or an acceptor fluorophore. In other embodiments, a label can be a redox reporter.
As used herein, the term “scaffold strand” refers to a component of the antibody construct (e.g., a dual-antibody construct) that does not have an antibody linked to it. In some embodiments, a scaffold strand can have one or more labels linked to the scaffold strand. In some embodiments, the scaffold strand can be a polynucleotide. In some embodiments, the scaffold strand can contain a mixture of nucleotides, amino acids, and small organic molecules. In certain embodiments, the scaffold strand can contain small organic molecules (e.g., polyethylene glycol (PEG) and/or polypropylene glycol (PPG)), in addition to nucleotides. The scaffold strand can be designed to provide flexibility to the conformation of the antibody construct. In some embodiments, one or more scaffold strands or portions thereof in an antibody construct are complementary and can hybridize to one or more detecting strands or portions thereof to form a double-stranded polynucleotide in an antibody construct.
As used herein, the term “detecting strand” refers to a component of the antibody construct that comprises an antibody and can recognize and bind to a target analyte via the affinity between the antibody conjugated to the detecting strand and the target analyte. In some embodiments, the full length or only a portion of the detecting strand is hybridized to one or more scaffold strands or portions thereof. In some embodiments, the antibody is conjugated to a terminus of the detecting strand. In other embodiments, the antibody is conjugated to an internal region of the detecting strand. In some embodiments, the detecting strand can be a polynucleotide. In some embodiments, the detecting strand can contain a mixture of nucleotides, amino acids, and small organic molecules. In certain embodiments, the detecting strand can contain small organic molecules (e.g., polyethylene glycol (PEG) and/or polypropylene glycol (PPG)), in addition to nucleotides.
As used herein, the term “antibody” refers to a protein functionally defined as a binding protein and structurally defined as comprising an amino acid sequence that is recognized by one of skill as being derived from a variable region of an immunoglobulin encoding gene. The term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, and human antibodies. The term “antibody,” as used herein, also includes antibody fragments that retain binding specificity, including but not limited to Fab, F(ab′)2, Fv, scFv, and bivalent scFv. An antibody can consist of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. The term “antibody,” as used herein, also includes antibody mimetic proteins, for example affibodies, DARPins, and nanobodies.
An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (VL) and “variable heavy chain” (VH) refer to these light and heavy chains, respectively.
As used herein, the term “detectable readout” refers to a measurable signal or the absence of any measurable signal. A measurable signal can be a physical and/or chemical signal, e.g., a fluorescent signal or an electrochemical signal. For example, a detectable readout can be a fluorescent signal produced by a fluorophore. A detectable readout can also be the disappearance of a fluorescent signal when the fluorophore is interacting with a quencher, which quenches the fluorescent signal.
As used herein, the term “fluorophore” refers to a compound, e.g., a small molecule or a protein, which when excited by exposure to a particular wavelength of light, emits light at a different wavelength. Fluorophores can be characterized in terms of their emission profile, or “color.” For example, green fluorophores (e.g., green fluorescent protein (GFP), Cy3, FITC, and Oregon Green) are generally characterized by their emission at wavelengths in the range of 510-550 nm. Red fluorophores (e.g., red fluorescent protein (RFP), Texas Red, Cy5, and tetramethylrhodamine) are generally characterized by their emission at wavelengths in the range of 590-690 nm.
As used herein, the term “target analyte” refers to a molecule that can be recognized and bound by the antibody in the antibody construct. A target analyte can be a small molecule (e.g., a small organic molecule), a protein, a glycan, a drug (for example but not limited to vancomycin), a peptide, or a nucleic acid (e.g., DNA or RNA).
As used herein, the term “blocking analyte” refers to a molecule that can be recognized and bound by the antibody in the antibody construct and is linked to the antibody by way of a linker. In some embodiments, the blocking analyte can be the same as the target analyte. In other embodiments, the blocking analyte can be a structural analog of the target analyte, which means that the blocking analyte can have a structure similar to that of the target analyte but differing from the target analyte in certain components of the structure, such as one or more atoms or functional groups. In yet other embodiments, a blocking analyte can be a molecule that can bind to the antibody in the antibody construct independent of the antibody's cognate antigen type.
As used herein, the term “hybridize” or “hybridization” refers to the annealing of nucleobases, nucleosides, or nucleotides of a polynucleotide to the nucleobases, nucleosides, or nucleotides at the corresponding positions of another polynucleotide. In some embodiments, hybridization can happen through hydrogen bonding interactions that occur between nucleobases, nucleosides, or nucleotides. The hydrogen bonding interactions may be Watson-Crick hydrogen bonding or Hoogsteen or reverse Hoogsteen hydrogen bonding. In some embodiments, nucleobases, nucleosides, or nucleotides of a polynucleotide can hybridize to the nucleobases, nucleosides, or nucleotides at the corresponding positions of another polynucleotide via partial complementarity or complete complementarity.
As used herein, the term “complementary” or “complementarity” refers to the capacity for base pairing via Watson-Crick hydrogen bonding interactions between nucleobases, nucleosides, or nucleotides of a polynucleotide to the nucleobases, nucleosides, or nucleotides at the corresponding positions of another polynucleotide. Examples of complementary nucleobase pairs include, but are not limited to, adenine and thymine, cytosine and guanine, and adenine and uracil, which all pair through the formation of hydrogen bonds. In some embodiments, a polynucleotide or a portion thereof in the antibody construct can have “complete complementarity” to another polynucleotide or a portion thereof, which means that all of the nucleotides the first polynucleotide that form the hybridization are complementary to the corresponding nucleotides in the other polynucleotide. In other embodiments, a polynucleotide or a portion thereof in the antibody construct can have “partial complementarity” to the other polynucleotide or a portion thereof, which means that at least one of the nucleotides in the first polynucleotide does not form Watson-Crick hydrogen bonding with the nucleotide at the corresponding position of the other polynucleotide. Two polynucleotides having “partial complementarity” can contain at least one mismatched nucleotide and can be referred to as a partially double-stranded polynucleotide. A “degree of complementarity” or “% complementarity” refers to a percentage of nucleobases, nucleosides, or nucleotides of a polynucleotide that form Watson-Crick hydrogen bonding interactions with the nucleobases, nucleosides, or nucleotides at the corresponding positions of the other polynucleotide. A “perfect degree of complementarity” or “100% complementarity” refers to that all of the of nucleobases, nucleosides, or nucleotides in the first polynucleotide or a portion thereof that form the hybridization with the other polynucleotide or a portion thereof are in Watson-Crick hydrogen bonding interactions with the nucleobases, nucleosides, or nucleotides at the corresponding positions of the other polynucleotide.
As used herein, the term “mismatched nucleotide” refers to a nucleotide at a specific position in the polynucleotide that does not engage in Watson-Crick base pairing with a nucleotide at the corresponding position in another polynucleotide when the two polynucleotides hybridize.
The disclosure provides antibody constructs that can change their conformation upon binding to a target analyte. An antibody construct can contain one antibody conjugated to a first label, and the antibody can be further linked to a blocking analyte and a second label by way of a linker. In another example, an antibody construct can be a dual-antibody construct that contains two antibodies linked to detecting strands. The detecting strands can further hybridize to one or more scaffold strands to form an antibody construct having the structure of a double-stranded or partially double-stranded polynucleotide.
An antibody construct disclosed herein can be a single-antibody construct. In one example, a single-antibody construct comprises: an antibody or a binding fragment thereof comprising a first label, wherein the antibody or the binding fragment thereof is linked to a blocking analyte and a second label via a linker. In a single-antibody construct, in the absence of a target analyte, the blocking analyte binds to the antibody or the binding fragment thereof, and in the presence of the target analyte, the target analyte competes with the blocking analyte for binding to the antibody or the binding fragment thereof. Further, the first and second labels in the single-antibody construct can interact to generate a detectable readout that changes depending on whether the blocking analyte is bound by the antibody or the binding fragment thereof, or is not bound by the antibody or the binding fragment thereof.
In a single-antibody construct, in some embodiments, the first label can be conjugated to the antibody and the second label can be conjugated to the linker. (see, e.g.,
The first and second labels are selected so that signal from the labels change based on their proximity, namely such that there is a measurable difference in signal when the antibody binds to the blocking analyte compared to a conformation when the blocking analyte is displaced by the target analyte. In certain embodiments, the first label can be a fluorophore and the second label can be a quencher. In certain embodiments, the first label can be a quencher and the second label can be a fluorophore. In cases where one of the two labels is a quencher, in the absence of a target analyte, the blocking analyte binds to the antibody, bringing the first and second labels in proximity, such that the fluorescent signal from one label is quenched by the other label and no signal is detected. In the presence of a target analyte, the target analyte can bind to the antibody, such that the first and second labels are not in proximity to each other and the fluorescent signal from one of the labels is detected.
In other embodiments, the first label can be a donor fluorophore and the second label can be an acceptor fluorophore to form a FRET pair. In other embodiments, the first label can be an acceptor fluorophore and the second label can be a donor fluorophore to form a FRET pair. Examples of fluorophores that form FRET pairs are described in detail further below. In the absence of a target analyte, the blocking analyte binds to the antibody, bringing the first and second labels in proximity, such that the emission spectrum of the donor fluorophore partially overlaps the excitation spectrum of the acceptor fluorophore and a FRET signal is produced. In the presence of a target analyte, the target analyte can bind to the antibody, such that the first and second labels are not in proximity to each other and no FRET signal is produced.
The detectable readout generated from the single-antibody construct can be an optical signal (e.g., a fluorescent signal), an electrical signal, an electrochemical signal, a nuclear magnetic resonance signal, or a biological signal. The binding of the target analyte to the antibody or the binding fragment thereof can increase or decrease the detectable readout.
In another example of a single-antibody construct, an antibody or a binding fragment thereof can be conjugated to a redox reporter and further linked to a blocking analyte and a sensing electrode via a linker. See, e.g.,
As shown in
As shown in
In certain embodiments, the sensing electrode can be a gold electrode. The linker can be engineered to contain a thiol functional group, which can form a gold-thiol bond between the linker and the gold electrode. Examples of redox reporters include, but are not limited to, methylene blue, thionine, anthraquinone, nile blue, gallocyanine, ferrocene, and pentamethyl ferrocene
In any of the single-antibody constructs described herein, the linker(s) can be a double-stranded polynucleotide or a single-stranded polynucleotide. In some embodiments, the linker can be a partially double-stranded polynucleotide comprising at least one unpaired nucleotide (e.g., between 1 and 20, between 1 and 18, between 1 and 16, between 1 and 14, between 1 and 12, between 1 and 10, between 1 and 8, between 1 and 6, between 1 and 4, between 1 and 3, between 2 and 20, between 4 and 20, between 6 and 20, between 8 and 20, between 10 and 20, between 12 and 20, between 14 and 20, between 16 and 20, or between 18 and 20 unpaired nucleotides; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 unpaired nucleotides). In some embodiments, the linker can be a partially double-stranded polynucleotide, in which at least 1% (e.g., between 1% and 20%, between 1% and 18%, between 1% and 16%, between 1% and 14%, between 1% and 12%, between 1% and 10%, between 1% and 8%, between 1% and 6%, between 1% and 4%, between 2% and 20%, between 4% and 20%, between 6% and 20%, between 8% and 20%, between 10% and 20%, between 12% and 20%, between 14% and 20%, between 16% and 20%, or between 18% and 20%) of the length of the linker comprises unpaired nucleotide(s). The length of the linker can be at least 10 nucleotides, e.g., between 10 and 100 nucleotides (e.g., between 10 and 90, between 10 and 80, between 10 and 70, between 10 and 60, between 10 and 50, between 10 and 40, between 10 and 30, between 10 and 20, between 10 and 15 nucleotides, between 20 and 100, between 30 and 100, between 40 and 100, between 40 and 80, between 50 and 80, between 50 and 100, between 50 and 70, between 60 and 100, between 70 and 100, between and 100, or between 90 and 100; e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, or 100 nucleotides).
In certain embodiments, the unpaired nucleotides can be located at the 5′ terminus and/or the 3′ terminus of the linker or in the middle of the linker sequence. Further, the linker can contain one or more mismatched nucleotides (e.g., between 1 and 20, between 1 and 18, between 1 and 16, between 1 and 14, between 1 and 12, between 1 and 10, between 1 and 8, between 1 and 6, between 1 and 4, between 1 and 3, between 2 and 20, between 4 and 20, between 6 and 20, between 8 and 20, between 10 and 20, between 12 and 20, between 14 and 20, between 16 and 20, or between 18 and 20) mismatched nucleotides; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mismatched nucleotides). The linker can also or alternatively contain one or more bulges, which are unpaired stretches of nucleotides located within one strand of a double-stranded or partially double-stranded polynucleotide. Bulge sizes can vary from a single unpaired nucleotide up to several nucleotides (e.g., between 2 and 20, between 2 and 18, between 2 and 16, between 2 and 14, between 2 and 12, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 4, between 4 and 20, between 6 and 20, between 8 and 20, between 10 and 20, between 12 and 20, between 14 and 20, between 16 and 20, or between 18 and 20 nucleotides). Moreover, a linker in an antibody construct described herein can also contain one or more nicks (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nicks) in one or both strands of a double-stranded or partially double-stranded polynucleotide linker. A linker described herein can also contain one or more non-natural nucleotides (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-natural nucleotides). Examples of non-natural nucleotides are described in, e.g., Stovall et al., Curr Protoc Nucleic Acid Chem 56:9.6.1-33, 2014; and Saito-Tarashima and Minakawa, Review Chem Pharm Bull (Tokyo), 66(2):132-138, 2018, and are also described in detail further herein. The presence of one or more mismatched nucleotides, bulges, nicks, non-natural nucleotides, and/or unpaired nucleotides can alter the flexibility and/or temporal sensitivity of the construct.
In a single-antibody construct described herein, in some embodiments, the blocking analyte and the target analyte can be the same. In some embodiments, the blocking analyte and the target analyte can be the different. In other embodiments, the blocking analyte can be a structural analog of the target analyte, in which one or more atoms or functional groups in the blocking analyte are different from the target analyte. For example, desthiobiotin is a structural analog of biotin and can be used as a blocking analyte in a single-antibody construct. In certain embodiments, a binding affinity of the antibody to the target analyte is between 90% and 110% (e.g., between 90% and 105%, between 90% and 100%, between 90% and 95%, between 95% and 110%, between 100% and 110%, or between 105% and 110%) of a binding affinity of the antibody to the blocking analyte. In certain embodiments, the antibody has a higher binding affinity for the target analyte than for the blocking analyte. In other embodiments, the antibody has a lower binding affinity for the target analyte than for the blocking analyte.
In some embodiments, a blocking analyte can be a molecule that comprises the antibody's cognate antigen, an epitope of the antibody's cognate antibody, or a fragment thereof. In some embodiments, a blocking analyte can be a molecule that can bind to the antibody in the antibody construct independent of the antibody's cognate antigen type. For example, a blocking analyte can be a protein that generally blocks antibody-antigen binding, for example, but not limited to, by binding to the antibody's conserved regions. A blocking analyte that binds to the antibody in the antibody construct independent of the antibody's cognate antigen type can be, e.g., Protein M (see, e.g., Grover et al., Science, 343(6171):656-661, 2014), Protein A, Protein G, and Protein L. In yet other embodiments, a blocking analyte that binds to the antibody in the antibody construct independent of the antibody's cognate antigen type can be a DNA aptamer or a nanostructure.
The antibody can be covalently conjugated to the linker using available conjugation techniques in the art (see, e.g., Gong et al., Bioconjugate Chem. 27(1):217-225, 2016). For example, the antibody can be covalently conjugated to the linker via its Fc region.
An antibody construct disclosed herein can be a dual-antibody construct. In one example, a dual-antibody construct can comprise: (a) two detecting strands, wherein a first detecting strand comprises a first antibody or a binding fragment thereof, and a second detecting strand comprises a second antibody or a binding fragment thereof; and (b) a first label and a second label, in which the detecting strands or portions thereof are complementary and hybridize to each other or one or more scaffold strands, and wherein in the presence of the target analyte, the first antibody and the second antibody binds to two different epitopes on the target analyte, and the first and second labels interact with each other to generate a detectable readout compared to when there is an absence of the target analyte.
In some embodiments of the dual-antibody construct, the first label is linked to the first antibody and the second label is linked to the second antibody. In other embodiments, the first label and/or the second label can be linked to one or more scaffold strands. For example, the first label and the second label can each be linked to a separate scaffold strand. In another example, the first label can be linked to one terminus of the scaffold strand and the second label can be linked to the other terminus of the same scaffold strand. In the presence of a target analyte, the first antibody and the second antibody can bind to different epitopes on the target analyte, bringing the first label and the second label in proximity of each other to generate a detectable readout.
In one example of a dual-antibody construct, the antibody construct can contain a scaffold strand and two detecting strands, wherein each of the two detecting strands or a portion thereof is complementary and hybridizes to the first scaffold strand or a portion thereof (see, e.g.,
Further, in some embodiments of the dual-antibody construct, the detecting strands and/or the scaffold strand(s) can comprise one or more mismatched nucleotides. The presence of one or more mismatched nucleotides can alter the flexibility and/or temporal sensitivity of the construct.
In some embodiments of the dual antibody aspect, one or both antibodies are linked directly to the labels (e.g., the first label is linked to the first antibody and the second label is linked to the second antibody). In these aspects, binding of the antibodies to a target analyte bring the first and second labels in proximity allowing for a change in detectable signal compared to in the absence of the target analyte.
In some embodiments, the first label and/or second label is linked to a polynucleotide strand (e.g., a “label oligonucleotide”) that hybridizes to a portion of the scaffold. See, e.g.,
In some embodiments, the first label and/or second label is linked to a polynucleotide strand that is part of the scaffold. In these embodiments, the labels are brought into proximity by the binding of the antibodies.
The first and second antibodies are each linked to a detecting polynucleotide strand. These detecting strands can have complementary ends allowing for them to directly hybridize. Alternatively, the polynucleotide strands can hybridize to one or more scaffold strands to indirectly link the two antibodies. For example,
An antibody construct described herein comprises labels that function to produce a detectable readout when the antibody construct is in a reporting state, i.e., when a target analyte is present. In some embodiments, the labels can produce a chemical and/or physical signal as a detectable readout when the antibody construct binds to a target analyte.
In some embodiments, the antibody construct generates fluorescence as a detectable readout when it binds to a target analyte. In some embodiments, one of the first and second labels on the antibody construct can be a fluorophore and the other of the first and second labels can be a quencher. In this case, the blocking analyte binds to the antibody in the absence of the target analyte and the fluorescence signal is quenched.
In some embodiments, one of the first and second labels on the antibody construct can be a donor fluorophore and the other of the first and second labels can be an acceptor fluorophore and the first and second labels can form a FRET pair. It is known that, in order for two fluorophores to be FRET partners, the emission spectrum of the donor fluorophore must partially overlap the excitation spectrum of the acceptor fluorophore. In some embodiments, the preferred FRET-partner pairs are those for which the value R0 (Förster distance, distance at which energy transfer is 50% efficient) is greater than or equal to 30Å. Examples of FRET partners are known in the art, see, e.g., Massey et al., Analytica Chimica Acta 568:181-189, 2006. In some embodiments, when the antibody is bound by the blocking analyte in the absence of a target analyte, the first and second labels are in proximity of each other to produce a FRET signal. The disappearance or reduction of the FRET fluorescence can serve as a signal of target analyte binding. In other words, in the absence of the target analyte, the fluorescent signal from the acceptor fluorophore can serve as the detectable signal. In the presence of the target analyte and the formation of the antibody-target analyte complex, the donor fluorophore and the acceptor fluorophore are not in proximity of each other to produce a FRET signal and the fluorescent signal of the donor fluorophore can serve as the detectable readout. Other fluorescence based methods that can be used to investigate the structure, binding, and dynamics of an antibody construct can be found in, e.g., Perez-Gonzales et al., Front Chem. 4:33, 2016.
Examples of fluorophores, as well as quenchers, are known in the art, e.g., as described in Marras, Methods Mol Biol. 335:3-16, 2006; Kozma and Kele, Org Biomol Chem. 17(2):215-233, 2019; and Wang et al., Angew Chem Int Ed Engl. Mar. 7, 2019. Efficient and complete quenching of the fluorescence emitted from the fluorophore by the quencher depends in part on the overlap between the fluorophore emission and quencher absorption spectra. For example, fluorophore coumarin emits at emission wavelength around 472 nm and can be paired with quencher QSY35 which absorbs at wavelength around 475 nm. In another example, fluorophore Alexa 532 emits at emission wavelength around 554 nm and can be a paired with quencher QSY7 which absorbs at wavelength around 560 nm. In yet another example, fluorophore Alexa 647 emits at emission wavelength around 665 nm and can be paired with quencher QSY21 which absorbs at wavelength around 661 nm.
In other embodiments, a label can be a fluorophore whose fluorescence can be quenched. An example of such a fluorophore is 2-amino purine, whose fluorescence can be quenched when it is stacked with purines and/or pyrimidines (see, e.g., Jean and Hall, Proc Natl Acad Sci USA. 98(1):37-41, 2001).
In other embodiments, the labels in the antibody construct can produce chemical and/or physical signals as a detectable readout when the antibody construct binds to a target analyte. These signals can be monitored to infer binding to the target analyte. In one example, the labels can be electrochemical reporters (see, e.g., Ferguson et al., Sci Transl Med. 5(213):213ra165, 2013). A first label can be an electrode (e.g., a gold electrode) and a second label can be a redox reporter (e.g., methylene blue). In some embodiments, upon binding to the target analyte, the antibody construct undergoes a conformational rearrangement that modulates the redox current and generates an electrochemical signal.
In some embodiments, the antibody constructs can include one or more non-natural nucleotides (e.g., non-natural DNAs and/or non-natural RNAs). A non-natural nucleotide can include one or more of a non-natural nucleobase, a non-natural sugar, and a non-natural internucleoside linkage.
A non-natural nucleobase refers to a nucleobase having at least one change that is structurally distinguishable from a naturally-occurring nucleobase (i.e., adenine, guanine, cytosine, thymine, or uracil). In some embodiments, a non-natural nucleobase is functionally interchangeable with its naturally-occurring counterpart. Both naturally-occurring and non-natural nucleobases are capable of hydrogen bonding. Modifications on non-natural nucleobases may help to improve the stability of the antibody constructs to nucleases. In some embodiments, an antibody construct described herein may include at least one non-natural nucleobase. Examples of non-natural nucleobases include, but are not limited to, 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propyladenine, 2-propylguanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-halouracil, 5-bromouracil, 5-trifluoromethyluracil, 5-halocytosine, 5-bromocytosine, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 2-fluoroadenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine. In some embodiments, an antibody construct described herein has one or more non-natural nucleobases (e.g., 5-methylcytosine).
A non-natural sugar refers to a sugar having at least one change that is structurally distinguishable from a naturally-occurring sugar (i.e., 2′-deoxyribose in DNA or ribose in RNA). Modifications on non-natural sugars may help to improve the stability of the antibody constructs to nucleases. In some embodiments, the sugar is a pentofuranosyl sugar. The pentofuranosyl sugar ring of a nucleoside may be non-natural in various ways including, but not limited to, addition of a substituent group, particularly, at the 2′ position of the ring; bridging two non-geminal ring atoms to form a bicyclic sugar (i.e., a locked sugar); and substitution of an atom or group such as —S—, —N(R)— or —C(R1)(R2) for the ring oxygen. Examples of non-natural sugars include, but are not limited to, substituted sugars, especially 2′-substituted sugars having a 2′—F, 2′—OCH2 (2′—OMe), or a 2′—O(CH2)2—OCH3 (2′—O—methoxyethyl or 2′—MOE) substituent group; and bicyclic sugars. A bicyclic sugar refers to a non-natural pentofuranosyl sugar containing two fused rings. For example, a bicyclic sugar may have the 2′ ring carbon of the pentofuranose linked to the 4′ ring carbon by way of one or more carbons (i.e., a methylene) and/or heteroatoms (i.e., sulfur, oxygen, or nitrogen). The second ring in the sugar limits the flexibility of the sugar ring and thus, constrains the polynucleotide in a conformation that is favorable for base pairing interactions with its target nucleic acids. An example of a bicyclic sugar is a locked sugar, which is a pentofuranosyl sugar having the 2′-oxygen linked to the 4′ ring carbon by way of a carbon (i.e., a methylene) or a heteroatom (i.e., sulfur, oxygen, or nitrogen). In some embodiments, a locked sugar has the 2′-oxygen linked to the 4′ ring carbon by way of a carbon (i.e., a methylene). In other words, a locked sugar has a 4′—(CH2)—O—2′ bridge, such as α-L-methyleneoxy (4′—CH2—O—2′) and β-D-methyleneoxy (4′—CH2—O—2′). A nucleoside having a lock sugar is referred to as a locked nucleoside.
Other examples of bicyclic sugars include, but are not limited to, (6′S)-6′ methyl bicyclic sugar, aminooxy (4′—CH2—O—N(R)—2′) bicyclic sugar, oxyamino (4′—CH2—N(R)—O—2′) bicyclic sugar, wherein R is, independently, H, a protecting group or C1-C12 alkyl. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), wherein each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
In some embodiments, a non-natural sugar is an unlocked sugar. An unlocked sugar refers to an acyclic sugar that has a 2′, 3′-seco acyclic structure, where the bond between the 2′ carbon and the 3′ carbon in a pentofuranosyl ring is absent.
An internucleoside linkage refers to the backbone linkage that connects the nucleosides. An internucleoside linkage may be a naturally-occurring internucleoside linkage (i.e., a phosphate linkage, also referred to as a 3′ to 5′ phosphodiester linkage, which is found in DNA and RNA) or a non-natural internucleoside linkage. A non-natural internucleoside linkage refers to an internucleoside linkage having at least one change that is structurally distinguishable from a naturally-occurring internucleoside linkage. Non-natural internucleoside linkages may help to improve the stability of the antibody constructs to nucleases and enhance cellular uptake.
Examples of non-natural internucleoside linkages include, but are not limited to, a phosphorothioate linkage, a phosphorodithioate linkage, a phosphoramidate linkage, a phosphorodiamidate linkage, a thiophosphoramidate linkage, a thiophosphorodiamidate linkage, a phosphoramidate morpholino linkage, and a thiophosphoramidate morpholino linkage, and a thiophosphorodiamidate morpholino linkage, which are known in the art and described in, e.g., Bennett and Swayze, Annu Rev Pharmacol Toxicol. 50:259-293, 2010. A phosphorothioate linkage is a 3′ to 5′ phosphodiester linkage that has a sulfur atom for a non-bridging oxygen in the phosphate backbone of a polynucleotide. A phosphorodithioate linkage is a 3′ to 5′ phosphodiester linkage that has two sulfur atoms for non-bridging oxygens in the phosphate backbone of a polynucleotide. A thiophosphoramidate linkage refers to a 3′ to 5′ phospho-linkage that has a sulfur atom for a non-bridging oxygen and a NH group as the 3′-bridging oxygen in the phosphate backbone of a polynucleotide. In some embodiments, an antibody construct described herein has at least one phosphorothioate linkage. In some embodiments, all of the internucleoside linkages in an antibody construct described herein are phosphorothioate linkages.
The disclosure also provides methods of detecting a target analyte in a sample (e.g., a biological sample), comprising: (1) contacting the sample with an antibody construct described herein; and (2) measuring binding of the antibody construct to the target analyte using a detectable readout (e.g., an optical signal, an electrical signal, an electrochemical signal, a nuclear magnetic resonance signal, or a biological signal) from the antibody construct.
Further, the disclosure also includes methods of adjusting kinetics and/or effective binding affinity of an antibody construct, comprising: (1) generating an antibody construct described herein; (2) measuring binding of the antibody construct to a target analyte; (3) changing one or more components of the antibody construct; (4) re-measure binding of the antibody construct to the target analyte; and (5) optionally repeat steps (3) and (4) until the desired kinetics and/or effective binding affinity of the antibody construct is reached. One component that can be tailored to adjust the kinetics and/or effective binding affinity of the antibody construct is the length and/or nucleotide composition of the linker. In some embodiments, increasing or decreasing the length of the linker can provide or remove flexibility and/or sensitivity of the antibody construct. Further, introducing or removing one or more mismatched nucleotides, bulges, nicks, non-natural nucleotides, and/or unpaired nucleotides from the linker can also provide or remove flexibility and/or sensitivity of the antibody construct. Another component that can be tailored to adjust the kinetics and/or effective binding affinity of the antibody construct is the identity of the blocking analyte. In some embodiments, changing the identity of the blocking analyte can vary the affinity of the antibody for the blocking analyte and thus the strength of intramolecular binding competition, or can vary the dissociation and association rates of the blocking analyte and thus the rate of target binding and blocking.
In a dual-antibody construct, increasing or decreasing the length of one or more scaffold strands provide or remove flexibility and/or sensitivity of the antibody construct. Furthermore, introducing or removing one or more mismatched nucleotides, bulges, nicks, non-natural nucleotides, and/or unpaired nucleotides from one or more scaffold strands can also provide or remove flexibility and/or sensitivity of the antibody construct.
The reversible, modular, and programmable nature of the antibody constructs enables the kinetics and the effective binding affinity of the antibody construct to be fine-tuned for the desired purpose and use of the construct, such as applications in molecular diagnostics, biomedical imaging, point-of-care devices, and home-use tests. Depending on the use format, the antibody construct can be modulated to have rapid, quantitative, sensitive, and multiplexed target analyte detection in complex systems.
The antibody switch construct depicted in
Antibody switch constructs depicted in
Antibody switch constructs depicted in
Antibody constructs depicted in
The antibody switch construct depicted in
The following discussion and
A fluorescently-reporting single antibody switch construct was synthesized from a vancomycin-specific monoclonal IgG and the vancomycin-DNA conjugate (60nt DNA) described above using the same process outlined in
A surface-tethered bead-based reversibility assay showed that the biotin-responsive single antibody switch (
A surface-tethered bead-based assay showed that the biotin-responsive single antibody switch responded to changing target concentration with fast temporal resolution that enables rapid measurement. The antibody switch construct (
The antibody-switch construct employs a single antibody and achieves target-responsive molecular switching in response to a small molecule steroid, digoxigenin (DIG). In contrast to the previous results which all demonstrated biotin-responsive behavior, this construct expands the design to function in sensing a new molecule. The design leverages the same architecture as shown in previous results, but replaces the anti-biotin monoclonal antibody with an anti-digoxigenin polyclonal antibody. The blocking strand, biotin in the previous embodiment, was replaced with digoxigenin. Surface-tethered bead-based measurements of concentration-dependent switching (
A surface-tethered bead-based reversibility assay showed that the digoxigenin-responsive single antibody switch can be used to continuously measure changing analyte concentrations through changing fluorescent output over many cycles of increasing or decreasing concentrations (
Protein detection is of great importance to clinical diagnostics as it is one of the predominant methods of detecting and monitoring disease states. Protein quantitation in blood samples is typically achieved through ELISA assays. Due to the sample preparation and assay protocols, these assays are typically performed in off-site or centralized analytical laboratories by specialized technicians—this involves lengthy turnaround times that can impact patient outcomes. Current and past research into point-of-care analytical systems seeks to address the need for sensitive, rapid, and sample-prep-free protein detection and quantitation that can be carried out in the clinical setting, thus reducing sample-to-answer times.
Certain proposed methods address these needs by adopting analyte-specific detection approaches, such as enzymatic sensors, which are not readily generalizable to a plurality of targets. Other approaches, such as PLA (proximity ligation) and ECPA (electrochemical proximity assay), leverage the generality of immunosorbent assays in homogeneous assays, yet still require sample processing, lengthy incubations and/or addition of reagents. Ideally, a point-of-care protein detection system would require the simple addition of a blood draw to a benchtop instrument which achieves rapid quantitation at relevant endogenous target concentrations.
Here, we propose a new approach to protein quantification which preserves the generality of immunosorbent techniques in an assay free of sample handling or processing steps. This is achieved with a monolithic dual antibody ‘clamp’ (MDAC) construct, an embodiment of a double-antibody construct, conjoined by a DNA scaffold. When the two fluorophore-labeled monoclonal antibodies bind and ‘clamp down’ on a single target protein, their proximity leads to a FRET signal change. Excitation and probing of the MDAC is achieved by surface coupling with a tapered fiber optic (fiber-MDAC): an evanescent excitation field probes the binding state of MDAC constructs and the FRET emission response couples back into the fiber for optical detection. The fiber optic tip is directly immersed in a blood draw sample and quantitation is achieved within minutes. The fiber could be easily disposed and replaced for subsequent measurements. The platform is adaptable to small protein targets with at least two binding domains, which includes any target for which a traditional sandwich ELISA assay exists, holding great promise as a new rapid immunosorbent analytical technique for the clinical environment.
The MDAC construct consists of two fluorophore-labeled monoclonal antibodies linked by a DNA scaffold (
First, we assembled MDAC on streptavidin beads (
After each assembly step we monitored the fluorescence of the beads with a flow cytometer (
The proximity of the two scaffold-bound antibodies warrants advantageous binding properties, among which is a strong avidity for the target. The binding behavior is approximated by a 5-state model (
In some cases, however, one of the two targets may be shared between the antibodies. This leads to FRET-competent state S2. The presence of two targets will affect the entropic state space associated with intramolecular binding—we model this difference with an adjustment factor α for Ceff. Boltzmann weights corresponding to the states described above are reported in Table 1, and the resulting expression for the total FRET-competent partition is given by,
Boltzmann weight multiplicities are due to the existence of two Fab regions on each antibody, which increases the possible binding configurations. For sake of simplicity, we chose to exclude further binding configurations, such as those involving three or more targets and those involving more than one shared target between the antibodies. These configurations will tend to appear at exceedingly high target concentrations or are expected to be comparatively unfavorable at the observed concentrations.
We validated the binding model experimentally by first measuring the affinity of clones mAb1 and mAb11 via biolayer interferometry (BLI) (
k
obs
=k
on
·T+k
off. (Eq. 2)
The dissociation constants, KD, of the antibodies were then computed via the definition
These were found to be 6.5 nM and 15.1 nM for mAb1 and mAb11, respectively. Next, we challenged MDAC beads with a wide range of unlabeled TNFa concentrations, allowed the structure to reach equilibrium overnight, and measured donor and FRET signals on a cytometer (
Next, we tested the feasibility of using MDAC to perform rapid quantitation of target concentrations directly in serum. MDAC constructs were assembled on beads and dispensed in chicken serum (CS) titrated with a range of TNFa concentrations. The beads were incubated for 30 minutes, magnetically separated from serum, washed, and resuspended in buffer. The procedure was repeated with TNFa in buffer as control. The beads were finally imaged on a cytometer to measure donor and acceptor fluorescence (
The data shows a significant increase in FRET fluorescence and decrease in green donor fluorescence between 100 pM and 1 nM, indicating that the binding kinetics of this MDAC structure are sufficiently rapid to carry out a measurement in only 30 minutes. For MDAC deployed in serum, we also note a significant decrease in green fluorescence at lower concentrations. This is attributed to the activity of nucleases in the serum that degrade the MDAC DNA scaffold, leading to the loss of the distal green antibody. The decrease in green donor fluorescence at low concentrations leads to an increase in FRET ratio background (
To further confirm that nuclease activity was the source of this signal degradation, we deployed the MDAC beads in target-free serum and characterized degradation of the structure over the course of 70 minutes (
To explore the generalizability of the platform towards different targets, a new MDAC structure was synthesized for MCP-1. For this iteration of the MDAC structure, Alexa Fluor 647 was substituted with Atto 643—although these two dyes are spectrally similar, Atto 643 has significantly higher quantum efficiency. Previous literature has shown that a high FRET acceptor quantum yield is of key importance to improved FRET efficiency, as a larger portion of the energy transferred from the donor dye is converted to observable fluorescence. Commercial vendors have reported that antibody clone 5D3-F7 forms sandwich pairs on MCP-1 targets both with clone 10F7 and clone 2H5. Bead MDAC structures were assembled with both pairs and preliminary binding curves in buffer showed that the 5D3-F7/10F7 structure resulted in larger FRET ratio signals, likely due to a more favorable placement of binding pockets—thus we proceeded with these clones for subsequent experiments.
As a first step, the assembly of the MCP-1 MDAC on beads was characterized, with controls for non-specific assembly with the new antibody clones (
Next, we verified binding performance of the bead-immobilized structure in CS doped with MCP-1 concentrations, with a limited incubation time of 30 minutes (
Optical fiber surfaces were prepared with MCP-1 MDAC and exposed to different concentrations of MCP-1 in buffer (
The MDAC is employed in an instant ELISA system through an optical fiber backend (
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application claims priority to U.S. Provisional Application No. 63/090,918, filed Oct. 13, 2020, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/054522 | 10/12/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63090918 | Oct 2020 | US |
