Patent Application
20230341395
The subject matter disclosed generally relates to genetic assemblies of inorganic and organic binding entities to functionalize various biosensors for the detection of any pathogens of interest.
Pathogen detection for many applications primarily relies on three different technologies: i) culture-based methods, ii) immunoassays (such as enzyme linked immunosorbent assay (ELISA)) and iii) polymerase chain reaction (PCR)-based methods. While cultures and ELISA are sensitive methods for pathogen detection, their main drawback is turnaround time with cultures taking days to generate a result. Although PCR is very sensitive, and faster than the culture-based methods and immunoassays, it requires technical expertise and a multi-step process to first isolate DNA or RNA for analysis. Furthermore, PCR is not able to differentiate between viable and nonviable pathogens.
Human coronaviruses are positive sense, single stranded RNA viruses. There are seven types of coronaviruses known to infect humans. Patients infected with these viruses develop respiratory symptoms of various severity. HCoV-229E and HCoV-0C43 are well known and cause common colds. Five other coronaviruses lead to more severe respiratory tract infection, which can potentially be lethal. Since 2000, there have been three major world-wide health crises caused by coronaviruses, the 2003 SARS outbreak, the 2012 MERS outbreak, and the most recent 2019 COVID-19 outbreak.
Biosensors, analytical devices that combine a biological component with a physiochemical detector for the detection of a chemical substance, can be categorized based on their capture elements (enzyme-based, immunosensors using antibodies, DNA biosensors, etc.), or their transducers (thermal, piezoelectric biosensors, etc.). The best-known biosensors are the lateral flow-based pregnancy test and the electrochemical glucose biosensors.
The immobilization of the capture elements or bioreceptors on the surface is of great importance as they not only functionalize but also determine the sensitivity of the biosensor. There are two groups of immobilization methods: irreversible and reversible. Irreversible immobilization includes covalent binding, cross-linking and entrapment, while reversible methods include random adsorption, bioaffinity (biotin/streptavidin and protein A/G), chelation/metal binding and disulfide bonds (LIÉBANA; DRAGO, 2016).
Antibodies are sensing biomolecules often used for the clinical application of biosensors. The easiest way of preparing a sensor with antibodies is random adsorption. Random adsorption, however, is associated with the denaturation of proteins, very low stability and random orientation, thus affecting the performance of the biosensor. The most widely used method for antibody immobilization is through covalent binding which, however, also results in random orientations of the antibodies as the amino/carboxyl groups used in the covalent bonds are uniformly distributed on the antibody.
There is a need in the art for improved biosensors. The present disclosure addresses this need by providing dual-affinity probes and biosensors for the detection of analytes, including but not limited to pathogens, with the sensitivity and specify needed in various applications, including in a point of care setting.
The disclosure provides dual affinity probes and related methods of use, e.g., to determine the presence of and/or amount or quantity of a target analyte in a sample. The dual affinity probes comprise: (i) an inorganic surface binding element, and (ii) a capture element.
According to an embodiment of the invention, there is provided a dual-affinity immunoprobe for detecting an analyte, e.g., a pathogen, in a sample, the immunoprobe including an inorganic surface binding peptide and an analyte-specific capture element. In embodiments, the analyte-specific capture element is an organic binding entity specific for the analyte, e.g., pathogen. In other embodiments, the capture element is selected from protein G from Streptococcus, streptavidin from Streptomyces, a single chain variable fragment, a Fab fragment, or an antibody. In particular embodiments, the capture element specifically binds to the analyte, e.g., pathogen.
In certain embodiments, the capture element is connected to the inorganic surface binding peptide via a linker sequence. In still other embodiments, the inorganic surface binding peptide binds specifically to a biosensor material selected from the group consisting of gold, silica, silver, cellulose (e.g., nitrocellulose), plastic, polystyrene, and graphene.
In embodiments, the analyte-specific capture element specifically binds the analyte. In embodiments, the analyte-specific capture element is a pathogen-specific capture element that specifically binds the pathogen. In some embodiments, the pathogen is SARS-CoV-2.
In embodiments of the invention, there is provided a dual-affinity probe wherein an inorganic surface binding peptide comprises gold-, silver,- silica-, plastic-, cellulose-, polystyrene-, or graphene-binding peptides fused to protein G or streptavidin, and a capture element comprises antibodies that specifically binds a target analyte.
In embodiments of the invention, there is provided a dual-affinity probe wherein an inorganic surface binding peptide comprises gold-, silver,- silica-, plastic-, cellulose-, polystyrene-, or graphene-binding peptides fused to protein G or streptavidin, and a capture element comprises S or N antigen targeting antibodies specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen.
In embodiments, the inorganic surface binding peptide is selected from Table 1 herein. In another embodiment, the inorganic surface binding peptide is selected from EMT014, EMT015, EMT016, EMT017, EMT018, EMT019, EMT020, EMT021, EMT022, EMT023, EMT024, EMT025. In another embodiment, the inorganic surface binding peptide is selected from cellulose binding motif 1, cellulose binding motif 2, polystyrene binding motif 1, polystyrene binding motif 2, and silica binding motif.
According to an embodiment, there is provided a platform using gold-binding peptides fused to protein G and coupled to S antigen targeting antibodies for detecting novel coronavirus SARS-CoV-2 via SARS-CoV-2 Spike (S) antigen in some embodiments, and Nucleocapsid (N) antigen in other embodiments.
According to another embodiment, there is provided a platform using silica-binding peptides fused to protein G and coupled to N antigen targeting antibodies for detecting novel coronavirus SARS-CoV-2 via SARS-CoV-2 Nucleocapsid (N) antigen.
According to yet another embodiment, there is provided a platform using gold-binding peptides fused to protein G and coupled to S and N antigen targeting antibodies for detecting novel coronavirus SARS-CoV-2 via SARS-CoV-2 Spike (S) antigen.
According to another embodiment, there is provided a platform using silica-binding peptides fused to protein G and coupled to S and N antigen targeting antibodies for detecting novel coronavirus SARS-CoV-2 via SARS-CoV-2 Nucleocapsid (N) antigen.
According to an embodiment, there is provided a platform using gold-binding peptides fused to streptavidin and coupled to S and N antigen targeting antibodies for detecting novel coronavirus SARS-CoV-2 via SARS-CoV-2 Spike (S) antigen in some embodiments, and Nucleocapsid (N) antigen in other embodiments.
According to an embodiment, there is provided a platform using cellulose-binding peptides, silica-binding peptides fused to streptavidin, or polystyrene-binding peptides which are then fused to streptavidin and coupled to S and N antigen targeting antibodies for detecting novel coronavirus SARS-CoV-2 via SARS-CoV-2 Spike (S) antigen in some embodiments, and Nucleocapsid (N) antigen in other embodiments.
In embodiments, the platform detects the pathogens via quartz crystal microbalance with dissipation (QCM-D). In other embodiments, the platform detects the pathogens via surface plasmon resonance (SPR). In still other embodiments, the platform detects the pathogens via lateral flow.
In a specific embodiment, the invention may be a dual-affinity probe for detecting an analyte, e.g., a pathogen, in a sample, the probe comprising a surface binding moiety (SBM), wherein the surface binding moiety is optionally an inorganic surface binding peptide (ISBP), and a capture element (CE). In a specific embodiment, the capture element (CE) is connected to the inorganic surface binding peptide via one or more linker (LI), wherein each LI may independently be a single bond or an amino acid sequence. In certain embodiments, the one or more linkers are passive linkers and/or active linkers. In a specific embodiment the probe has the following formula (I) or formula (II):
SBM-LI-CE (Ia) or CE-LI-SBM (IIa).
The capture element CE may be an organic binding entity specific for the analyte, wherein the analyte is optionally a pathogen or a fragment thereof. In a specific embodiment, the capture element comprises an antibody or an antigen-binding fragment thereof, optionally a single chain variable fragment (scFv) or a Fab fragment; or an antigen.
In another embodiment, the LI comprises one or more linkers, wherein each linker is independently a single bond, such as an ionic or covalent or non-covalent bond, or is selected from one or more of the group consisting of: a peptide or amino acid linker, an amino acid sequence comprising protein G from Streptococcus, and an amino acid sequence comprising streptavidin from Streptomyces. In another embodiment, LI comprises or is the protein G from Streptococcus or streptavidin from Streptomyces.
In another embodiment, the SBM or ISBP binds specifically to a biosensor material selected from the group consisting of gold, silica, silver, cellulose, plastic, polystyrene and graphene. In a further embodiment, the biosensor material is selected from the group consisting of gold, cellulose, silica and polystyrene.
In a more specific embodiment, the SBM or ISBP is selected from the group consisting of a binding peptide, a protein, an antibody with an affinity to the inorganic surface, or an immunogenic fragment thereof, optionally a single chain variable fragment (scFv) or a Fab fragment. In a specific embodiment, the SBM or ISBP is a binding peptide. In another embodiment, the ISBP is selected from the group consisting of any peptide sequence of Table 1 herein.
In another embodiment, the SBM or ISBP is an antibody, a single chain variable fragment from an antibody, or a Fab fragment. In a specific embodiment, the SBM or ISBP comprises a gold binding motif. In a further specific embodiment, the gold binding motif is a VH gold binding motif. In another embodiment the SBM or ISBP is an antibody. In a more specific embodiment, the SBM or ISBP is an antibody specific to binding gold.
In another embodiment of the dual-affinity probes, the CE is an antibody, or an antigen-binding fragment thereof, optionally an scFv or a Fab. In a specific embodiment, the CE is an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated with biotin, and the LI is an amino acid sequence comprising streptavidin from Streptomyces. In another specific embodiment, the CE is an antibody or an antigen-binding fragment thereof, and the LI is an amino acid sequence comprising protein G from Streptococcus. In a specific embodiment, the CE is an S or N antigen targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen, or an antigen-binding fragment thereof.
In another specific embodiment, the CE is an antigen. In another specific embodiment, the CE is an antigen fused to a linker or SBM/ISBP. In another specific embodiment, the CE antigen is biotinylated and binds to a streptavidin linker. In another specific embodiment, the CE is an antigen that binds to an antibody (or antibodies), wherein the antibody or antibodies are the intended analyte for detection. In a specific embodiment, the antigen protein is SARS-CoV-2 Spike and/or SARS-CoV-2 Nucleocapsid proteins. In another specific embodiment, the antigen binds to and detects antibodies. In another embodiment, the antibody or antibodies are a targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen, or an antigen-binding fragment thereof. In another embodiment of the dual-affinity probes, LI is a single bond, such as a covalent bond, or a peptide or amino acid linker. In a specific embodiment, the amino acid linker is a passive linker to allow, for example, space between the CE and ISBP, or to provide some rigidity or flexibility to the CE and SBM or ISBP combination. In a specific embodiment, the dual-affinity probe is a single fusion protein. In another embodiment, the CE and the SBM or ISBP is independently an antibody, or an antigen-binding fragment thereof, optionally a single chain variable fragment. In a specific embodiment, the ISBP is the single chain variable fragment. In a more specific embodiment, the single chain variable fragment is a VH gold binding motif. In another embodiment, the CE is a single chain variable fragment from an antibody. In a more specific embodiment, the SBM or ISBP and the CE are fused as a bispecific antibody fragment. In a specific embodiment, the SBM or ISBP is a single chain variable fragment that is a VH gold binding motif, and the CE is a single chain variable fragment specific to an antigen. In another embodiment, one or both of the CE and the SBM or ISBP is an antibody. In a specific embodiment, the CE and the ISBP are fused to form a bispecific immunoglobulin A. In a specific embodiment, the ISBP is specific for gold, silica, silver, cellulose, plastic, polystyrene, or graphene. In a further specific embodiment, the ISBP is specific for gold. In another embodiment, the CE is specific to an antigen of SARS-CoV-2. In a specific embodiment, the CE is specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen. In another embodiment, the CE is an S or N antigen targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen, or an antigen-binding fragment thereof.
The present invention also includes composition comprising one or more dual-affinity probes. In particular embodiments, the compositions are liquid compositions, wherein the dual affinity probes are present, e.g., in a buffered solution. In other embodiments, the compositions are solid compositions to which one or more dual affinity probes are bound or immobilized on.
The present invention may also include dual-affinity probes incorporated into a specific system or diagnostic system, such as for a specific point of care diagnostic system. Any diagnostic system comprising a dual-affinity probe may be used. For example, in a specific embodiment, the system includes analysis performed on a quartz crystal microbalance, a surface plasmon resonance (SPR), and/or performed via lateral flow. In a specific embodiment, the system is used for the detection of an analyte, e.g., a pathogen, of known sequence, comprising a dual-affinity probe. In a specific embodiment, the dual-affinity probe may be any probe described herein. The system may for example include any dual-affinity probe bound to an inorganic surface biosensor material selected from the group consisting of gold, silica, silver, cellulose, plastic, and graphene. In a specific system, the dual-affinity probe capture element is specific for SARS-CoV-2 (Spike or Nucleocapsid) protein.
The present invention also comprises methods of analyte, e.g., pathogen, detection using dual-affinity probes to analyze a medium for an analyte, e.g., a pathogen. In a specific embodiment, the dual-affinity probes may be any dual-affinity probe described herein. In another embodiment of the methods, the analysis is performed on a quartz crystal microbalance with dissipation (QCM-D), using surface plasmon resonance (SPR), and/or performed via lateral flow.
In a specific embodiment of the methods of the present invention, the method includes determining the presence of and/or quantifying an analyte, e.g., a pathogen, in a test sample, comprising:
In a specific embodiment of the methods, the test sample is a biological sample obtained from a subject. In a specific embodiment, the subject is a mammal, optionally a human. In another embodiment, the biological sample comprises serum, plasma, whole blood, saliva, mucus, nasal fluid, cerebrospinal fluid, sweat, urine or a combination thereof. In another embodiment, the analyte is a pathogen. In a specific embodiment, the pathogen is a virus, a bacterium, a fungi, a protozoa, a worm, or a prion. In a specific embodiment, the virus is a SARS-CoV-2 virus. In a further specific embodiment, the analyte-specific capture element comprises antibodies, or antigen-binding fragments thereof, specific for a SARS-CoV-2 Spike (S) antigen or a SARS-CoV-2 Nucleocapsid (N) antigen.
In another embodiment of the methods, the inorganic surface binding polypeptide comprises one or more gold-, silver-, silica-, plastic-, cellulose- or graphene- binding peptides. In another embodiment, the inorganic surface binding polypeptide comprises a peptide selected from any peptide sequence of Table 1 herein. In another embodiment, the dual-affinity probe is bound to surface, such as an inorganic surface. In another embodiment, the surface is a biosensor material selected from the group consisting of gold, silica, silver, cellulose, plastic, and graphene. In a specific embodiment of the methods, the specific contacting and/or determining is performed using a quartz crystal microbalance, surface plasmon resonance (SPR) or via lateral flow.
Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:
It will be noted that throughout the appended drawings, like features are identified by like reference numerals.
The following terms are defined below.
As used herein, the term “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including but not limited to scFv, Fab, and Fab2, so long as they exhibit the desired biological activity.
“Antibody fragments” comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., a capture element of a dual affinity probe) is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
The term “antigen-binding fragment” as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chain, or of a Nanobody® (Nab), that binds to the antigen of interest, e.g., a pathogen. In this regard, an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL from antibodies that bind one or more analyte, e.g., pathogen.
The term “a linker sequence” is intended to mean a sequence that bridges the surface binding entity, e.g., inorganic surface binding entity, with the organic binding entity. E.g., capture element. As used herein, a linker sequence may comprise one or both of an active linker and/or a passive linker. Thus, a linker sequence may, for example, comprise the amino acid sequence of protein G from Streptococcus or streptavidin from Streptomyce, or may be a simple amino acid sequence or simply a single bond, such as a covalent bond. Organic binding entities include both synthetic carbon-based compounds as well as biologically-derived molecules.
The term “surface binding motif” or SBM is intended to mean a molecule with specific and selective affinity for an organic or inorganic substance, such as, e.g., gold, silica, silver, plastic, polystyrene, cellulose (e.g., nitrocellulose), and graphene. An SBM may be a peptide or polypeptide. The term “inorganic surface binding peptides” or ISBP is intended to mean a sequence of amino acids with specific and selective affinity for an inorganic substance such as gold, silica or graphene. The ISBP may thus, for example, include a short peptide, a protein, an antibody with an affinity to the inorganic surface or fragment of an antibody, such as a single chain variable fragment (scFv).
The term “biosensor” is intended to mean a component or device that converts the detection of an analyte, e.g., a pathogen, into a measurable signal using biological components. The term “biosensor material ” is intended to mean something that converts biological or chemical reactions into measurable signals that are proportional to an analyte, e.g., a pathogen, of interest. The signal generated can be in the form of heat, light, pH, mass or charge change, for example.
The term “capture element” is intended to include an antigen, protein G from Streptococcus or streptavidin from Streptomyces or a single chain variable fragment or a Fab fragment or an antibody, for example SARS-CoV-2 Spike and SARS-CoV-2 Nucleocapsid targeting antibodies. “Capture elements” include any moiety capable of binding to the analyte or target being detected and/or quantified.
The term “covalent fusion” is intended to mean the joining of two or more genes that encode separate peptides or proteins. The terms “polypeptide” “protein” and “peptide” are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms “polypeptide” or “protein” or “peptide” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein or peptide can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. Thus, a “polypeptide” or a “protein” can comprise one (termed “a monomer”) or a plurality (termed “a multimer”) of amino acid chains.
The term “fusion protein” means a protein comprised of at least two different amino acid sequences and generated within an organism such as E. coli or insect cells of Spodoptera frugiperda. An inorganic surface binding peptide expressed with A or G protein or a linker is an example of a fusion protein.
“Pathogens” include pathogenic agents that cause mammalian infection or disease, including, e.g., viruses, bacteria, etc., such as any of those disclosed herein, including but not limited to: SARS-CoV-2, influenza viruses, Adenovirus, CMV, Coxsackievirus, Dengue Virus, Epstein Barr virus (EBV), Enterovirus 71 (EV71), Ebola Virus, Hepatitis A virus (HAV), Hepatitis B virus (HBV), Human cytomegalovirus (HCMV), Hepatitis C virus (HCV), Hepatitis D virus (HDV), Hepatitis E virus (HEV), Human Immunodeficiency Virus (HIV), Human papilloma virus (HPV), Herpes simplex virus (HSV), Human T-lymphotropic virus (HTLV), Influenza A Virus, Influenza B Virus, Japanese Encephalitis, Leukemia Virus, and Ebola Virus, Measles Virus, Molluscum Contagiosum, Orf Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Varicella Zoster Virus, Variola, West Nile Virus, Zika Virus, and Chikungunya Virus. The term “pathogen” is also intended to include proteins or peptides of a pathogen, including but not limited to proteins or peptides that indicate the presence of a disease-causing organism or virus, and/or biomarkers for a disease-causing organism or virus, for example spike and nucleocapsid proteins of human coronaviruses, including SARS-CoV-2, influenza hemagglutinin, antigens of Adenovirus, CMV, Coxsackievirus, Dengue Virus, EBV, EV71, Ebola Virus, HAV, HBV, HCMV, HCV, HDV, HEV, HIV, HPV, HSV, HTLV, Influenza A Virus, Influenza B Virus, Japanese Encephalitis, Leukemia Virus, Measles Virus, Molluscum Contagiosum, Orf Virus, Parvovirus, Rabies Virus, Respiratory Syncytial Virus, Rift Valley Fever Virus, Rubella Virus, Rotavirus, Varicella Zoster Virus, Variola, West Nile Virus, Zika Virus, and Chikungunya Virus.
The term “specifically binds” means that a molecule reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target molecule, e.g., a pathogen, than it does with alternative molecules, e.g., pathogens. It is also understood by reading this definition that, a molecule that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding.
With respect to antibodies, KD is the equilibrium dissociation constant, a calculated ratio of Koff/Kon, between the antibody and its antigen. The association constant (Kon) is used to characterise how quickly the antibody binds to its target. The dissociation constant (Koff) is used to measure how quickly an antibody dissociates from its target. KD and affinity are inversely related. A high affinity interaction is characterized by a low KD, a fast recognizing (high Kon) and a strong stability of formed complexes (low Koff). In certain embodiments, a dual affinity probe, or the capture element thereof binds to its target with a KD of at least or less than 1×102, at least or less than 1×103, at least or less than 1×104, at least or less than 1×105, at least or less than 1×106, at least or less than 1×107, at least or less than 1×108, at least or less than 1×109, at least or less than 1×1010, at least or less than 1×1011, or at least or less than 1×1012. For purposes of this invention, KD is determined from a binding curve using a Biacore2000 measuring device according to the analysis software provided with the device.
Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.
In this disclosure, the word “comprising” is used in a non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
It will be understood that in embodiments which comprise or may comprise a specified feature or variable or parameter, alternative embodiments may consist, or consist essentially of such features, or variables or parameters. A reference to an element by the indefinite article “a” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements.
In this disclosure the recitation of numerical ranges by endpoints includes all numbers subsumed within that range including all whole numbers, all integers and all fractional intermediates (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc). In this disclosure the singular forms an “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds.
In this disclosure term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
The disclosure provides compositions and methods for detecting the presence and or quantity of an analyte in a test sample.
Aspects of the disclosure related to dual-affinity probes, or specifically dual-affinity immunoprobes that may be used to determine the presence or absence or an analyte in a test sample, wherein the dual-affinity probes comprise an inorganic surface binding polypeptide and an analyte-specific capture element.
In a specific embodiment, the compositions may comprise a dual-affinity probe, which may be use for detecting an analyte, e.g., an infectious agent or pathogen, in a sample, the dual-affinity probe comprising a surface binding motif (SBM), e.g., an inorganic surface binding peptide (ISBP), and a capture element (CE). In another embodiment, the dual-affinity probe may be a dual-affinity immunoprobe, meaning the probe may be utilized with the use of an antibody or antibody fragment. For example, the SBM and/or the CE may comprises an antibody or an antigen-binding fragment thereof.
In certain embodiments of dual-affinity probes, the SBM or ISBP is a peptide. In particular embodiments, the SBM or ISBP is an antibody, or an antigen-binding fragment thereof, e.g., an scFv. A variety of surface binding peptides are known in the art, and illustrative surface binding peptides are disclosed herein.
In particular embodiments, the analyte is a pathogen, and the analyte-specific capture element specifically binds to the pathogen. In certain embodiments, the analyte-specific capture element is an antibody, or an antigen binding fragment thereof, e.g., an scFv. Antibodies that specifically bind to various pathogens, including but not limited to those disclosed herein, are known in the art, and may be readily produced.
The disclosure contemplates various formats of dual-affinity probes. In certain embodiments, the dual-affinity probe comprises one or more polypeptide that binds to both a specific surface and one or more specific target analyte. In other embodiments, the dual-affinity probe comprises two or more polypeptides, including a first polypeptide that binds to a specific surface and also includes an active linker that binds to a class of molecules, such as antibodies, or to a specific member of a binding pair, such as streptavidin/biotin; and a second polypeptide comprising a target specific capture element, wherein the second polypeptide is bound by the active linker. For example, the second polypeptide may comprises an antibody, or antigen-binding fragment thereof, that specifically binds the target analyte, and/or it may comprise a member of a binding pair that is bound by the other member of the binding pair present in the first polypeptide. Thus, while certain dual-affinity probes specifically bind one or more target analytes, e.g., pathogens, other dual-affinity probes may be adapted to identity any of a variety of different target analytes, depending on the nature of the capture element, i.e., the target analyte it binds. Diagrams of various illustrative configurations of dual-affinity probes are provided in
In particular embodiments, the SBM and the CE are present within the same polypeptide, and may be directly fused to each other or fused to each other via one or more linker, e.g., a passive linker, such as a bond or a glycine-serine linker, or an IgA J chain or a llama IgG hinge region. In particular embodiments, the analyte-specific capture element specifically binds to an analyte of interest. In certain embodiments, the analyte-specific capture element is an antibody or an antigen-binding fragment thereof, e.g., such as an scFv. In certain embodiments, the dual-affinity probe is a single fusion protein. In another embodiment, the CE and ISBP is independently an antibody, a fragment of an antibody, or a single chain variable fragment from an antibody. In another embodiment, the ISBP is a single chain variable fragment from an antibody. In another embodiment, the single chain variable fragment is a VH binding motif. In a specific embodiment, the VH binding motif is a gold VH binding motif. In another embodiment, the CE is a single chain variable fragment from an antibody. In a specific embodiment, the ISBP and CE are fused as a bispecific antibody fragment.
In particular embodiments, the SBM and the CE are present in different polypeptides. For example, in certain embodiments, the dual-affinity probes comprise a first polypeptide comprising the SBM and an active linker, and a second polypeptide comprising the CE, wherein the active linker is capable of binding to the second polypeptide comprising the analyte-specific capture element. In certain embodiments, the active linker directly binds the analyte specific capture element; for example, the active linker may be protein A, protein G, or anti-IgG (e.g., goat anti-human IgG), and the analyte specific capture element may be an antibody, or antigen binding fragment thereof. In other embodiments, the analyte specific capture element is fused to a non-specific binding element that directly binds to the active linker; for example, the non-specific capture element may be biotin, and the active linker may be streptavidin, or vice versa. In certain embodiments, protein G is fused to the N-terminus or the C-terminus of the SBM, e.g., via a passive linker, such as a peptide linker. In certain embodiments, streptavidin is fused to the N-terminus or the C-terminus of the SBM, e.g., via a passive linker, such as a peptide linker. Various other binding pairs, in addition to biotin and streptavidin are known in the art, and could alternatively be used.
In certain embodiments, the capture element (CE) is connected to the SBM via a linker sequence (LI), wherein LI may be a single bond or an amino acid sequence, and the linker sequence is further connected to the SBM, e.g., an ISBP. In particular embodiments, the linker (LS) comprises one or more passive linker (PL) and/or one or more active linker (AL). The dual-affinity probe may have the following formula (I) or formula (II):
SBM-LI-CE (I)
CE-LI-SBM (II).
In certain embodiments, the dual-affinity probe comprises at least two polypeptides, including a first polypeptide of formula (IIIa) or (IIIb), wherein PL is a passive linker, such as a single bond or passive peptide linker, and AL is an active linker that binds to the polypeptide of formula IV(a) or (IVb), wherein active linker binder (ALB) is a polypeptide sequence bound by the AL, wherein LI is a passive linker, such as a single bond or passive peptide linker, and wherein ALB and AL may be absent or present:
SBM-PL-AL (IIIa)
AL-PL-SBM (IIIb)
ALB-PL-CE (IVa)
CE-PL-ALB (IVb).
In a specific embodiment, the SBM or ISBP is connected to an inorganic surface, which may include an inorganic surface of a biosensor or other biosensor material. The inorganic surface or biosensor material that the SBM or ISBP may be connected to may include, e.g., gold, silica, silver, cellulose, plastic, polystyrene and graphene. In a specific embodiment, the biosensor material is selected from the group consisting of gold, cellulose, silica and polystyrene.
The dual-affinity probes may use such materials in various forms of biosensors or diagnostic platforms. For example, the biosensors or platforms may use technologies such as quartz crystal microbalance, surface plasmon resonance (SPR) or by a lateral flow assay.
The dual-affinity probes may incorporate any SBM or ISBP or LI or CE in any combination as described herein.
The capture element (CE) of the present invention may include any organic binding entity that binds to a specific analyte of interest. In particular embodiments, the analyte is an infectious agent or pathogen, and the analyte-specific capture element specifically binds to the infectious agent or pathogen. In certain embodiments, the analyte-specific capture element is an antibody, or an antigen binding fragment thereof, e.g., an scFv. Antibodies that specifically bind to various infectious agents and pathogens, including but not limited to those disclosed herein, are known in the art, and may be readily produced. In a specific embodiment, the capture element a fragment of an antibody such as a single chain variable fragment, or a Fab fragment.
The capture element may also be an amino acid sequence that is not an antibody or antibody fragment, but any amino acid sequence, peptide, protein or specific antigen that binds to the analyte. In certain embodiments, the methods disclosed herein may be used to determine the presence and/or amount of antibodies that bind to an infectious agent or pathogen, including but not limited to any of those disclosed herein, present in a sample, e.g., a biological sample. In certain embodiments, the capture element may be applied to test the sample of the subject to determine if the subject has antibodies for a specific pathogen or infectious agent, and more specifically a specific antigen or epitope thereof that identifies the pathogen. Thus, in a specific embodiment, the capture element comprises at least a portion of an antigen, or epitope thereof, bound by one or more antibodies that specifically bind the pathogen. In certain embodiments, the antigen may be any agent capable of inducing an immune response, e.g., in a mammal, that results in the product of antibodies that bind the antigen.
The capture element may be specific to any analyte or pathogen of interest, for example, the capture element may be specific to an antigen, protein, peptide, nucleic acid or other organic element that identifies that a subject may be positive for or infected with a specific pathogen. In a specific embodiment the capture element is specific to an antigen for SARS-CoV-2. In another specific embodiment, the capture element is specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen. In a specific embodiment, the capture element is an antibody and is an S or N antigen targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen. In another specific embodiment, the antibodies may be the specific antibodies listed in Table 2 herein.
Other pathogens that the capture element may be specific for include, but are not limited to, Coronavirus spp. Such as SARS and MERS; Influenza spp.; Respiratory Synctial Virus spp.; Adenovirus spp.; Parainfluenza spp.; Filoviridae such as Ebola and Marburg; Hantavirus spp.; Arenaviridae such as Lassa; Bunyaviridae such as Rift Valley and Crimean-Congo; and Paramyxoviridae such as Hendra and Nipah; for example. Pathogens include, in some embodiments, prions. Pathogens include, in some embodiments, Gram negative and Gram positive bacteria. Other pathogens may include for example infectious diseases. The capture element for example may be specific to an analyte or antigen in infectious diseases such as hepatitis B & C, HIV, syphilis, chlamydia and gonorrhea.
In another embodiment, the capture element is an antigen and is specific to unique pathogen such as SARS-CoV-2. In a specific embodiment, the antigen comprises at least a portion of the spike protein of SARS-CoV-2. In another embodiment, the antigen comprises at least the full sequence of the spike protein or any variants thereof.
In another specific embodiment, the capture element (CE) is an antigen that is fused or bound to the dual affinity probe. In another specific embodiment, the CE is an antigen fused to a linker or SBM/ISBP. In another specific embodiment, the CE antigen is biotinylated and binds to a streptavidin linker. In another specific embodiment, the CE is an antigen that binds to an antibody (or antibodies), the intended analyte for detection. In a specific embodiment, the antigen protein is SARS-CoV-2 Spike and/or SARS-CoV-2 Nucleocapsid proteins. In another specific embodiment, the antigen binds to and detects antibodies. In another embodiment, the antibody or antibodies are a targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen, or an antigen-binding fragment thereof.
In a specific embodiment, the capture element may be linked to the linker (LI) or ISBP to ensure that there is effective binding to the analyte of interest. For example, the spike protein of SARS-CoV-2 may be linked to the linker (LI) or ISBP to ensure that the correct portion of the protein or epitope is exposed to the analyte, and in this case antibodies that would be specific to various portions of the spike protein. Methods for attaching a capture element or specific amino acid sequence to another amino acid sequence are known in the art, and may be applied in the specific invention described herein. For example, in another embodiment the capture element may be tagged or modified for the purpose of binding specifically to a linker or directly to the ISBP. For example, the capture element may be biotinylated solely for binding to a streptavidin linker, such as streptavidin from Streptomyces. In another embodiment, the capture element may be an antibody or an element that is modified to more efficiently bind to a linker such as protein G, which is specific to IgG and protein G from Streptococcus.
Linkers may be included in the dual affinity probes of the present invention. Linkers may include any appropriate amino acid sequence required to control steric hindrance and/or chemical interactions with sensor components (organic or inorganic materials, peptides and proteins, cross-linking reagents, etc.).
The linker sequences of the dual-affinity probes of the present invention may include one or more passive linkers and/or active linkers. In certain embodiments, a dual-affinity probe comprises a passive linker fused to an active linker, e.g., to link the SBM or ISBP to the active linker. As used herein, a passive linker does not specifically bind to a capture element or other polypeptide, and are typically present between two polypeptide sequences to control steric hindrance, e.g., to retain activity of the two linked polypeptides. In particular embodiments, a passive linker may be a single bond or an amino acid sequence that links the SBM or ISBP to the CE (or polypeptide comprising the CE). A passive linker may also be present between a CE and a member of a binding pair to which it is fused. The link may be a covalent bond, an ionic bond, a non-covalent bond such as with the use of high-affinity molecules.
As used herein, an active linker may be fused to the SBM or ISBP and specifically binds to a CE or a polypeptide comprising the CE (e.g., a member of a binding pair present in the polypeptide comprising the CE), and may be present to functionally link the SBM or ISBP to the CE. In particular embodiments, an active linker binds to antibodies or antigen-binding fragments thereof (e.g., human antibodies or fragments thereof). In certain embodiments, an active linker is a member of a binding pair, such as streptavidin/biotin. The link may be a covalent bond, an ionic bond, a non-covalent bond such as with the use of high-affinity molecules.
In another embodiment, the linker sequence may include other amino acid sequences, such as passive linkers, a linear tandem repeat polypeptides, a linear non-repeating polypeptides or linkers that allow for additional flexibility or rigidity to the SBM, ISBP or CE.
In a specific embodiment, the high affinity molecule in the linker (i.e., the AL) may be an amino acid sequence comprising protein G from Streptococcus, or an amino acid sequence comprising streptavidin from Streptomyces. In another embodiment, the linkers may include an additional AL to directly and covalently bond to the SBM, ISBP but with a high affinity to IgG or biotin incorporated in the capture element.
In a specific embodiment, the passive linker may include a glycine-serine linker, for example the following amino acid sequence:
The passive linker of SEQ ID NO: 1 may be further incorporated or fused with another amino acid sequence on the linker, e.g., an AL, such as a high affinity protein such as streptavidin or protein G. In a specific embodiment, SEQ ID NO: 1 is directly fused to protein G to form the following sequence [SEQ ID NO: 2] as follows:
In this example, the passive linker SEQ ID NO: 1 is on the C terminus of the AL and directly links to the SBM or ISBP, wherein the protein G amino acid sequence binds with high affinity to the capture element, which would be any IgG antibody or appropriate fragment of an IgG antibody.
In another specific embodiment, a passive linker such as SEQ ID NO: 1 may be fused to streptavidin (AL) in the linker. In a specific embodiment, the passive linker SEQ ID NO: 1 is on the C terminus of the AL and directly links to the SBM or ISBP, wherein the streptavidin amino acid sequence binds with high affinity to the biotinylated capture element.
In a specific embodiment, SEQ ID NO: 1 is directly fused to streptavidin to form the following sequence [SEQ ID NO: 21] as follows:
In this example, the passive linker SEQ ID NO: 1 is on the C terminus of the streptavidin AL and directly links to the SBM or ISBP, wherein the streptavidin amino acid sequence binds with high affinity to the capture element (or a polypeptide comprising the CE), which may be a biotinylated protein, including an antibody or antibody fragment.
In a specific embodiment, the ISBP fuse to the linker may be an amino acid sequence or peptide that binds to gold, silicon, cellulose, polystyrene, or silica. In another specific embodiment, the ISBP may be or comprise any one of SEQ ID NO: 3-19 or 25.
In another embodiment, no passive linker is included in the linker sequence. For example, the linker AL, may be specific to just the protein G amino acid sequence or the streptavidin amino acid sequence. In a specific embodiment, the linker (AL) may comprise the following sequence of protein G, [SEQ ID NO: 19] as follows:
In a specific embodiment, the linker (AL) is SEQ ID NO: 19.
In another embodiment, the linker (AL) may comprise the following sequence of streptavidin [SEQ ID NO: 22] as follows:
In a specific embodiment, the linker sequences may be there own fusion protein, or may incorporate other elements of the present invention, such as the SBM or ISBP and/or CE to form a fusion protein. Fusion proteins, including the design, gene synthesis, the cloning, expression, and purification thereof are known in the art, and can be incorporated to form any fusions thereof. For example, the linkers of the present invention may incorporate such sequences with tags for protein purification, such as His tags or other protein tags known in the art. The Examples of the present application provide examples of specific fusion proteins, but is not limiting to the invention herein.
In another specific embodiment, the LI linker may just be a single bond, such as a covalent bond. In such an example, the SBM or ISBP and CE are thus directly bonded to each other with no additional amino acid or atom representing the Linker.
The dual-affinity probes of the present invention may include a surface binding moiety (SBM) that binds to an organic or inorganic surface of choice. For example, the SBM binds specifically to a biosensor material selected from the group consisting of gold, silica, silver, cellulose, e.g., nitrocellulose, plastic, polystyrene and graphene. In particular embodiments, the SBM is an organic or inorganic surface binding polypeptide (ISBP). As used herein the ISBP may bind to organic or inorganic surfaces. In another example, the ISBP may bind specifically to a biosensor material selected from the group consisting of gold, cellulose, silica and polystyrene.
In a specific embodiment, the SBM or ISBP may include an amino acid sequence and may be selected from the group consisting of a binding peptide, a protein, an antibody with an affinity to the inorganic surface, or an antigen-binding fragment thereof, such as a single chain variable fragment (scFv). In particular embodiments, the inorganic surface binding polypeptide is a peptide. In particular embodiments, the inorganic surface binding polypeptide is an antibody, or an antigen-binding fragment thereof, e.g., an scFv. A variety of surface binding peptides are known in the art, and illustrative surface binding peptides are disclosed herein.
In a specific embodiment, the ISBP comprises a peptide specific to binding gold, cellulose, silicon or polystyrene. In another embodiment, the ISBP comprises a peptide from Table 1 provided herein.
In another embodiment, the ISBP comprises an antibody or a fragment of an antibody. In a specific embodiment, the ISBP is a VH or VL binding motif. In a specific embodiment, the ISBP is a gold VH or VL binding motif. In a specific embodiment, the antibody or a fragment of an antibody may be specific to binding gold. In a specific embodiment, the ISBP may be a gold-binding protein from U.S. Pat. No. 7,807,391, Shiotsuka et al., which is incorporated by reference herein in its entirety.
The dual-affinity probe of the present invention may have the following formula (Ia): ISBP-LI-CE (Ia) or formula (IIa): CE-LI-ISBP (IIa).
In a specific embodiment, capture element CE is an organic binding entity specific for the pathogen. The capture element is selected from a single chain variable fragment, a Fab fragment, an antibody, or an antigen; LI is a linker sequence comprising one or more passive linker and/or active linker. In certain embodiments, one or more of the linkers present in LI comprises a single bond, or is selected from one or more of the group consisting of an amino acid linker, an amino acid sequence comprising protein G from Streptococcus, or an amino acid sequence comprising streptavidin from Streptomyces; and the ISBP binds specifically to a biosensor material selected from the group consisting of gold, silica, silver, cellulose, plastic, polystyrene and graphene.
In a specific embodiment, LI is single bond, therein allowing ISBP to bind directly to CE.
In this arrangement, the dual affinity probes may comprise the inorganic surface binding polypeptide and the analyte-specific capture element within the same polypeptide, and may be directly fused to each other or fused to each other via one or more linker, e.g., a passive polypeptide linker. In particular embodiments, the analyte-specific capture element specifically binds to an analyte of interest. In certain embodiments, the analyte-specific capture element is an antibody or an antigen-binding fragment thereof, e.g., such as an scFv.
In a specific embodiment, the dual-affinity probe is a single fusion protein. In another embodiment, the CE and ISBP is independently an antibody, a fragment of an antibody, or a single chain variable fragment from an antibody. In another embodiment, the ISBP is a single chain variable fragment from an antibody. In another embodiment, the single chain variable fragment is a VH binding motif. In a specific embodiment, the VH binding motif is a gold VH binding motif. In another embodiment, the CE is a single chain variable fragment from an antibody. In a specific embodiment, the ISBP and CE are fused as a bispecific antibody fragment.
In a specific combination, the ISBP is a single chain variable fragment that is a VH gold binding motif, and the CE is a single chain variable fragment specific to an antigen.
In another specific combination, the CE and ISBP are each an antibody. In a specific embodiment, the CE and ISBP are fused to form a bispecific immunoglobulin A. In a specific embodiment, the CE and ISBP are fused to form a bispecific antibody fragment. In a specific embodiment, the CE and ISBP are fused to form a bispecific antibody fragment wherein the CE and ISBP or independently a VL fragment, VH fragment and/or a scFv fragment.
In another specific embodiment, the ISBP is specific for gold, silica, silver, cellulose, plastic, polystyrene and graphene. In a specific embodiment, the ISBP is specific for gold.
In another specific embodiment, the CE is specific to an antigen for SARS-CoV-2.
In a specific embodiment, the CE is specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen. In another specific embodiment, the CE is an antibody and is an S or N antigen targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen.
In another specific combination, the CE and ISBP are each an antibody with a linker in between. In a specific embodiment, the CE and ISBP are fused to form a bispecific immunoglobulin A. In a specific embodiment, the CE and ISBP are fused to form a bispecific antibody fragment. In a specific embodiment, the CE and ISBP are fused to form a bispecific antibody fragment wherein the CE and ISBP or independently a VL fragment, VH fragment and/or a scFv fragment.
In another specific embodiment, the CE is an antigen. In another specific embodiment, the CE is an antigen fused to a linker or SBM/ISBP. In another specific embodiment, the CE antigen is biotinylated and binds to a streptavidin linker. In another specific embodiment, the CE is an antigen that binds to an antibody (or antibodies), the intended analyte for detection. In a specific embodiment, the antigen protein is SARS-CoV-2 Spike and/or SARS-CoV-2 Nucleocapsid proteins. In another specific embodiment, the antigen binds to and detects antibodies. In another embodiment, the antibody or antibodies are a targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen, or an antigen-binding fragment thereof.
The dual-affinity probe of the present invention may comprise one or more polypeptide having the formula (IIIc) or (IIId) and one or more polypeptide having the formula (IVc) or (IVd):
ISBP-PL-AL (IIIc)
AL-PL-ISBP (IIId)
ALB-PL-CE (IVc)
CE-PL-ALB (IVd),
wherein LI, AL, and ALB are as defined for formulas (IIIa) and (IVa), and wherein PL may be present or absent from either or both the polypeptide of formula (IIIc) or (IIId) and/or the polypeptide of formula (IVc) or (IVd).
In a specific embodiment, PL comprises an amino acid sequence in between ISBP and CE. In particular embodiments, the AL if the polypeptide of formula (III) and the ALB of the polypeptide of formula (IV) are capable of binding to each or are bound to each other.
In such an arrangement, the inorganic surface binding polypeptide and the analyte-specific capture element may be present in different polypeptides. For example, in certain embodiments, the dual-affinity probes comprise a first polypeptide comprising the inorganic surface binding polypeptide and an active linker (AL), and a second polypeptide comprising the analyte-specific capture element, wherein the AL is capable of binding to the analyte-specific capture element (or a polypeptide comprising the CE). In certain embodiments, the AL directly binds the analyte specific capture element; for example, the AL may be protein A, protein G, or anti-IgG (e.g., goat anti-human IgG), and the analyte specific capture element may be an antibody, or antigen binding fragment thereof. In other embodiments, the analyte specific capture element is fused to a binding element (ALB) that directly binds to the AL; for example, the ALB may be biotin, and the AL may be streptavidin, or vice versa. In certain embodiments, protein G is fused to the N-terminus of the inorganic surface binding polypeptide, e.g., via a passive linker, such as a direct bond or a peptide linker. In certain embodiments, streptavidin is fused to the N-terminus of the inorganic surface binding polypeptide, e.g., via a passive linker, such as a direct bond or a peptide linker. In certain embodiments, protein G is fused to the C-terminus of the inorganic surface binding polypeptide, e.g., via a passive linker, such as a direct bond or a peptide linker. In certain embodiments, streptavidin is fused to the C-terminus of the inorganic surface binding polypeptide, e.g., via a passive linker, such as a direct bond or a peptide linker. Various other binding pairs, in addition to biotin and streptavidin are known in the art, and could alternatively be used.
In a specific embodiment, the ISBP of the dual-affinity probes is selected from the group consisting of a binding peptide, a protein, an antibody with an affinity to the inorganic surface, or an antigen-binding fragment thereof, such as a single chain variable fragment. In a specific embodiment, the ISBP is a binding peptide. In a specific embodiment, the binding peptide is from Table 1 herein.
In another specific embodiment, the ISBP is an antibody, a single chain variable fragment from an antibody or a Fab fragment. In a specific embodiment, the ISBP has a gold binding motif. In another specific embodiment, the ISBP is a VH binding motif. In another specific embodiment, the ISBP is a VH gold binding motif. In another specific embodiment, the ISBP is an antibody specific to binding gold.
In a further specific embodiment, AL is an amino acid sequence comprising protein G from Streptococcus or an amino acid sequence comprising streptavidin from Streptomyces.
In another embodiment, the linker sequences may include other amino acid sequences, such as passive linkers, a linear tandem repeat polypeptides, a linear non-repeating polypeptides or linkers that allow for additional flexibility or rigidity to the ISBP or CE.
In another embodiment, the linker sequences may include an additional passive linker to directly and covalently bond to the ISBP but with a high affinity to IgG or biotin incorporated in the capture element.
In a specific embodiment, the passive linker may include for example the following amino acid sequence:
The passive linker of SEQ ID NO: 1 may be further incorporated or fused with another amino acid sequence on the linker such as a high affinity protein such as streptavidin or protein G (AL). In a specific embodiment, SEQ ID NO: 1 is directly fused to protein G to form the following sequence [SEQ ID NO: 2] is:
In this example, the passive linker SEQ ID NO: 1 is on the C terminus and directly links to the ISBP, wherein the protein G amino acid sequence binds with high affinity to the capture element, which would be any IgG antibody or appropriate fragment of an IgG antibody.
In another specific embodiment, a passive linker such as SEQ ID NO: 1 may be fused to streptavidin in the linker. In a specific embodiment, the passive linker SEQ ID NO: 1 is on the C terminus and directly links to the ISBP, wherein the streptavidin amino acid sequence binds with high affinity to the biotinylated capture element.
In another embodiment, no passive linker is included in the linker sequences. For example, the linker AL, may be specific to just the protein G amino acid sequence such as SEQ ID NO: 19, variants thereof, or the streptavidin amino acid sequence.
In another specific embodiment, the CE is an antibody. In another specific embodiment, the CE is a fragment of an antibody. In a specific embodiment, and the antibody is conjugated with biotin (ALB), and the AL is an amino acid sequence comprising streptavidin from Streptomyces. In another embodiment, the CE is an antibody and the AL is an amino acid sequence comprising protein G from Streptococcus. In another embodiment, the CE is an antibody and is an S or N antigen targeting antibody specific for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen.
In various embodiments, the dual-affinity probes or immunoprobes are labeled with a detectable label. In particular embodiments of the dual-affinity immunoprobes, the polypeptide comprising the analyte-specific capture element is labeled with a detectable label.
The disclosure also provides a method of determining the presence of and/or quantifying an analyte in a test sample, comprising:
In some embodiments, the test sample is a biological sample, such as a biological sample obtained from a subject, such as, e.g., serum, plasma, whole blood, saliva, mucus, nasal fluid, nasopharyngeal secretions, middle ear fluid, cerebrospinal fluid, sweat, urine or a combination thereof. In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the biological sample comprises pathogens, antibodies, cells, and/or other biological molecules. The method may be used to test a variety of different types of samples, including, e.g., environmental samples (including samples collected in the built environment), water, or food or beverage samples, etc.
Methods of the disclosure may be used to assay for a variety of different analytes in a test sample. Examples of analytes include, but are not limited to, infectious agents, pathogens, antibodies that bind pathogens, specific cells, proteins, or carbohydrates, In certain embodiments, the analyte is an infectious agent or pathogen, and in certain embodiments, the infectious agent or the pathogen is a virus, a bacterium, a fungi, a protozoa, a worm, or a prion. In particular embodiments, the virus is an influenza virus or a coronavirus, e.g., SARS-CoV-2 virus. In other embodiments, the analyte is an antibody that specifically binds to one or more infectious agent or pathogen.
The methods may also use a capture element that is an amino acid sequence that is not an antibody or antibody fragment, but any amino acid sequence, peptide, protein or specific antigen that binds to an antibody from the pathogen. For example, the capture element may be used to test a biological sample obtained from a subject to determine if the subject has antibodies for a specific pathogen, and more specifically a specific antigen or epitope that identifies the pathogen. In a specific embodiment, the capture element comprises an antigen or epitope thereof. For example, a biotinylated SARS-CoV-2 Spike protein antigen may be conjugated to the streptavidin fusion protein for the detection of Spike protein specific antibodies in test samples.
The capture element may be specific to any analyte or pathogen of interest, for example, the capture element may be specific to an antigen, protein, peptide, nucleic acid, antibody or antibodies, or other organic element that identifies that a subject may be positive for or infected with a specific pathogen. In certain embodiments, the capture element is specific for an antibody that specifically binds an analyte or pathogen of interest. In a specific embodiment the capture element comprises an antigen for SARS-CoV-2. In another specific embodiment, the capture element comprises for SARS-CoV-2 Spike (S) antigen or SARS-CoV-2 Nucleocapsid (N) antigen or a variant thereof.
In various embodiments, the analyte-specific capture element specifically binds to an analyte of interest, in order to determine whether it is present in the test sample and/or the amount or concentration present in the test sample. In particular embodiments, the analyte-specific capture element comprises antibodies, or antigen-binding fragments thereof, specific for a pathogen or an antigen thereof, e.g., a SARS-CoV-2 Spike (S) antigen or a SARS-CoV-2 Nucleocapsid (N) antigen.
In particular embodiments, the inorganic surface binding peptide comprises one or more gold-, silver,- silica-, plastic-, cellulose- or graphene-binding peptides, including but not limited to any of the peptides of Table 1 herein.
In certain embodiments, the dual-affinity immunoprobe is bound to an inorganic surface via the inorganic surface binding peptide, and the test sample when the test sample is contacted with the dual-affinity immunoprobe. One example is a lateral flow assay. However, in other embodiments, the dual-affinity immunoprobe is not bound to the inorganic surface when the test sample is contacted with the dual-affinity immunoprobe. For example, the dual-affinity immunoprobe and the test sample may be contacted in a solution and form complexes, and the solution is then contacted with the inorganic surface, such that the dual-affinity immunoprobes to bind to the inorganic surface. In particular embodiments, the inorganic surface is a biosensor material selected from the group consisting of gold, silica, silver, cellulose, plastic, and graphene. Bound complexes or analyte may be detected and/or quantified via various means, for example using quartz crystal microbalance, surface plasmon resonance (SPR), or lateral flow.
In various embodiments, the methods may employ the use of one or more positive or negative control, e.g., a positive control test sample, a negative control test sample, and/or a negative control dual-affinity immunoprobe, an analyte-specific capture element that does not bind the analyte of interest.
In particular embodiments, the analyte is determined to be present in the test sample if it is detected in the test sample, or if a certain level or amount is determined to be present in the test sample. For example, the level or amount that indicates the presence of the analyte in the test sample may be a predetermined amount based on prior experience, or it may be an amount greater than the amount determined using a negative control, e.g., an amount at least 10%, at least 20%, at least 50%, at least two-fold, or an amount at least three-fold greater than the amount determined for a negative control.
In a specific embodiment, this detection of an analyte, i.e, confirmation of the subject being positive with the analyte, may determined by a binding curve, such as by SPR or QCM-D. In other words, the analyte is determined to be present such as obtaining a certain RU or other response or detection curve. In another embodiment, the analyte is determined to be present by a contrast from the negative control in color. Such contrast can be determined by visual determination of individual as instructed in the directions of the assay. Such determination can be performed in a point of care, hospital, or other healthcare facility. In another embodiment, the analyte is determined to be present by a contrast from the negative control in color by a device, such as a multiwell plate color reader.
The accompanying Examples are illustrative regarding certain specific embodiments of the compositions and methods disclosed herein.
Oriented loading of antibodies onto inorganic binding entity was achieved in one embodiment by adsorbing it to protein A and G, which contain binding domains for the Fc (Fragment crystallizable) region of antibodies.
In other embodiments, directed immobilization of recognition biomolecules (e.g., capture elements) is accomplished using the streptavidin-biotin system, which shows one of the strongest non-covalent interactions in nature.
In another embodiment, fusion proteins containing the inorganic binding peptide were linked to a single chain variable fragment (scFv) or a Fab fragment or a full-length antibody for the pathogen of interest. These methods may be employed in engineering dual-affinity immunoprobes of the invention. Other methods of reversibly and irreversibly binding antibodies and known in the art and are set out in detail in (MAKARAVICIUTE; RAMANAVICIENE, 2013) and (LIÉBANA; DRAGO, 2016).
Inorganic surface binding peptides may include those that specifically bind to gold, silica and graphene, as well as cellulose, silver, and carbon based synthetic polymers (plastics).
Sensor types may include planar gold, silver, and silica; gold and silver nanoparticles (nanoclusters, nanorods, etc . . . ); graphene sheets and tubes; cellulose sheets and strips; etched plastic sheets and slides, for example. Biosensor material includes gold, silver, silica, graphene, cellulose, and carbon based synthetic polymers, for example.
Pathogens may include Coronavirus spp. Such as SARS and MERS; Influenza spp.; Respiratory Synctial Virus spp.; Adenovirus spp.; Parainfluenza spp.; Filoviridae such as Ebola and Marburg; Hantavirus spp.; Arenaviridae such as Lassa; Bunyaviridae such as Rift Valley and Crimean-Congo; and Paramyxoviridae such as Hendra and Nipah; for example. Pathogens include, in some embodiments, prions. Pathogens include, in some embodiments, Gram negative and Gram positive bacteria.
Antibody types may include but are not limited to humanized, monoclonal, polyclonal, and synthetic antibodies.
Detection methods using the dual-affinity immunoprobes of the invention include but are not limited to lateral flow, in multiwell plate color readers; dipstick color change, SPR and Quartz crystal microbalance with dissipation monitoring (QCM-D).
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope
Acronyms or short forms used in the Examples
H=hours
Min=minutes
s=seconds
PBS=Phosphate Buffered Saline
E. coli=Escherichia coli
SARS-CoV-2=severe acute respiratory distress coronavirus 2
BSA=Bovine Serum Albumin
ddH2O=double distilled water
Identification and Synthesis of Synthetic peptides: Six gold-binding and six silica-binding peptides from the literature were contract synthesized with a purity of >90% using FMOC (Fluorenylmethyloxycarbonyl chloride) synthesis (Pierce ThermoFisher).
Design of fusion proteins: The general structure of the embodiments of the invention is inorganic surface binding peptide plus linker plus protein G′, a known version of protein G where the albumin binding site has been removed (a version of Uniprot Q54181 protein.). The Amino acid sequence of this linker plus protein G′ [SEQ ID NO: 2] is:
Antibodies and antigens: Monoclonal antibodies against the SARS-CoV-2 Spike protein (A02038), SARS-CoV-2 Nucleocapsid protein (A02039), and recombinant Spike (Z03501) and Nucleocapsid (Z03488) protein antigens, were purchased from Genscript (Piscataway, NJ).
Quartz crystal microbalance with dissipation monitoring (QCM-D) for comparative peptide binding analysis:
The Quartz Crystal Microbalance with dissipation monitoring (QCM-D) is an instrument that measures mass and viscosity in at or near surfaces and thin films. QCM-D can detect extremely small chemical, mechanical, and electrical changes taking place on a sensor surface, and convert them into electrical signals which can be interpreted (TONDA-TURO; CARMAGNOLA; CIARDELLI, 2018).
All QCM-D analyses were performed at 23° C. on the 4-channel Qsense™ Analyzer instrument (Biolin Scientific, Gothenburg, Sweden). The gold and silica Qsense™ sensor chips were rinsed in 70% ethanol, rinsed with deionized water, dried with compressed nitrogen, and then exposed to UV/ozone for 10 min to remove remaining organic residues. Samples were diluted to 100 μg/mL in 10 mM of PBS. The gold or silica sensor chips were loaded into the instrument and equilibrated for 15 min. Ten mM PBS was then flowed at 50 μL/min until an equilibrium for frequency and dissipation D Afn was attained.
The respective gold-binding and silica-binding peptides were flowed over their respective gold and silica sensors for 1 h, followed by a 10 mM PBS wash step for 30 min. The raw data was analyzed using Qsense™ Dfind™ analysis software using a Kelvin-Voigt viscoelastic model.
Surface Plasmon Resonance (SPR) analysis:
Surface Plasmon Resonance occurs when polarized light hits a metal film at the interface of media with different refractive indices. SPR techniques excite and detect collective oscillations of free electrons, by which light is focused onto a metal film through a glass prism and the reflection is detected. At a certain incident angle (or resonance angle), the electrons (aka plasmons) are set to resonate, resulting in absorption of light at that angle. This creates a dark line in the reflected beam.
The resonance angle can be determined by observing the SPR reflection intensity. A shift in the reflectivity curve represents a molecular binding event taking place on or near the metal film, or a conformational change in the molecules bound to the film. The shift vs. time provides information about molecular binding events and binding kinetics.
All SPR experiments were performed on an 8-channel Biacore™ 8K instrument (Cytiva Lifesciences (was GE Healthcare Lifesciences)), Marlborough, MA, USA) at 25° C. using the 2×HBS-EP+ running buffer and chips from the BiocoreTM SIA AU kit (Cytiva Lifesciences).
Generation and purification of gold-binding and silica-binding fusion proteins in Escherichia coli: The protein sequences for the fusion proteins, containing well described gold-binding (BROWN, 1997) and silica-binding (ETESHOLA; BRILLSON; LEE, 2005) peptides fused to a linker and the protein G′ protein from Streptococcus, were converted to cDNA using codon usage specific for E.coli. An N-terminal 6×histidine tag to the proteins were added for purification purposes. The cDNA inserts representing the fusion proteins were cloned in frame into the E. coli pET-30a (+) expression vector. Standard molecular cloning techniques were applied to identify the correct clones for protein expression. (SAMBROOK; FRITSCH; MANIATIS, 1989) The recombinant proteins were isolated from the supernatant of 1L expression cultures following a four-step purification protocol including Ni column, TEV protease digestion, Ni column and finally Q Sepharose column (all reagents from Genscript, Piscataway, NJ). The purity of the proteins was estimated by densitometric analysis of a Coomassie Blue-stained SDS-PAGE gel, and endotoxin levels were assessed using the LAL Endotoxin Assay Kit (Xiamen Bioendo Technology Co., Ltd., Xiamen, Fujian, China).
Purities of 90% were achieved for the fusion protein and the ISBP-free G′ proteins, as shown in
Functionalizing the QCM-D gold sensor with gold-binding fusion protein, and testing using the SARS-CoV-2 Spike protein antibody antigen system: Sensor chips were prepared and equilibrated in PBS as described above. Samples were diluted to 50 μg/mL using 10 mM PBS. The gold-binding fusion protein from Example 2 at 50 μg/mL in PBS was flowed over the sensor chips at 50 μL/min until Afn equilibrated, after which the sensor chips were washed with PBS followed by a BSA (50 μg/mL PBS) blocking step.
The SARS-CoV-2 Spike protein antibody was then flowed over the sensor chips at 50 μL/min, followed by a PBS wash step, and then finally the SARS-CoV-2 Spike antigen (50 μg/mL) or the negative control (SARS-CoV-2 Nucleocapsid antigen, 50 μg/mL) were flowed until the samples were consumed. The sensors were washed with PBS buffer to eliminate nonspecific binding. The raw data was analyzed in Qsense™ Dfind™ analysis software using a Kelvin-Voigt viscoelastic model.
The gold-binding fusion protein was found to bind to the gold sensor surface in two experiments, forming a 10.56 nm and 10.5 nm layer, respectively, with only a very small fraction washed off during the subsequent wash step (remaining layer thickness 9.66 nm and 9.6 nm, respectively). No significant changes to the thickness or mass of the layers occurred during the subsequent blocking with BSA and washing steps. The SARS-CoV-2 Spike protein antibody was then flowed across the biolayer and the thickness and mass of both layers more than doubled. After a second washing with PBS, a biolayer of 20.45 nm (
Evaluating the binding kinetics of the SARS-CoV-2 Spike antibody binding to the gold-binding fusion protein, and its ability to bind the Spike antigen: Surface plasmon resonance (SPR), an opto-electronic biosensing technique, was chosen to evaluate the binding kinetics of the Spike antibody to the gold-fusion protein bound to a gold sensor. First, the immobilization of the gold-binding fusion protein and two controls (ISBP-free fusion protein and buffer only) was evaluated. Zero or minimal binding was observed for those controls (
The ability and the binding kinetics of the SARS-CoV-2 Spike and Nucleocapsid antibodies to bind to the gold-binding fusion protein immobilized on the sensor surface, and the respective antigens binding to the antibodies, was tested using a dilution series. Dilution series for both antibodies (
In Table 2, the binding kinetics of the Spike protein antibody and the Nucleocapsid antibody to the gold-binding fusion protein are shown. The kinetics of interaction was calculated and dissociation constants (KD) of 1.92E-10 M and 2.58E-10 M were found for the SARS-CoV-2 Spike and Nucleocapsid antibody, respectively. This compares favorably to the KD levels reported in the literature which show that protein G binds all human IgG subclasses at ˜2E-10 M. As with QCM-D, these results show that the gold-binding fusion proteins efficiently bind to the gold sensor surface, immobilize and orient the SARS-CoV-2 Spike and Nucleocapsid antibodies. The SARS-CoV-2 S protein antigen then also binds to the Spike protein antibody with a KD of 2.39E-9 M, a typical range for a monoclonal antibody/antigen interaction, indicating that the bound Spike protein antibody was able to maintain its antigen binding affinity (
Conjugation of gold-binding fusion proteins to gold nanoparticles: The conjugation of the gold-binding fusion protein and the ISBP-free fusion protein control to 40 nm gold nanoparticles (Cytodiagnostics) was tested in 10 mM PBS buffer using increasing amounts of proteins (0, 1, 2, 4 μg per 100 μL of 1 OD gold) and increasing pH conditions (5.7-9.8). Results are shown in
Scale-up conjugation reaction for gold-binding fusion protein: The pH of 1 mL 40 nm standard gold nanoparticles was adjusted through the addition of 40 μL of 0.1M sodium phosphate pH 6.5. A 10 μg aliquot of fusion protein was transferred to a separate microcentrifuge vial and diluted to a total volume of 100 μL with ddH2O. The pH-adjusted gold nanoparticles were rapidly added to the vial of diluted fusion protein and incubated for 30 minutes at room temperature. 50 μL of 10% (w/v) BSA were added to the gold-fusion protein mixture and incubated for 5 minutes to block. The conjugation mixture was centrifuged at 1600×g for 25 minutes and the supernatant removed. Finally, the gold conjugate pellet was resuspended with 1×PBS, 1% BSA to a final concentration of OD=5.5 and stored at 4 degrees until use.
Comparative binding analysis of synthetic peptides to gold and silica sensors using QCM-D: Six gold-binding and six silica-binding peptides, described in the literature as binding to gold and silica and depicted in Table 1, were synthesized. Their ability to bind to gold and silica sensors was tested using quartz crystal microbalance with dissipation monitoring (QCM-D). The thickness, the mass deposited, elasticity and viscosity of the resulting layers after a PBS wash were calculated and are summarized in Table 4.
Table 4 summarizes comparative binding experiments of six gold-binding dual-affinity probes (EMT014-EMT019) and six silica-binding dual-affinity probes (EMT020-EMT025) using quartz crystal microbalance with dissipation monitoring (QCM-D). The mass (ng/cm2), molar mass μmol/m2), thickness (nm), elasticity (kPa) and viscosity (mPa s) for all peptides is reported.
EMT015, the longest gold-binding peptide, showed the highest mass (ng/cm2) deposited on the gold sensor, while EMT019, the shortest gold-binding peptide showed the highest loading when adjusted for the molecular weight of the peptide (indicated as molar mass (μmol/m2)). The adjusted measurement is a better indicator of the degree of binding. EMT015 built the thickest layer at 5.152 nm with EMT019 the second highest at 4.48 nm. The layer formed with EMT015 also showed higher elasticity and viscosity compared to the other peptides. For the silica-binding peptides, EMT022 showed the highest mass and molar mass deposited onto the silica sensor with a thickness of 4.1 nm compared to the other peptides. It also showed the highest viscosity and second highest elasticity.
Dot blot dipstick assay: Immobilization of antibodies onto gold-binding fusion protein coated gold nanoparticles and their antigen binding capacity was tested using a dot blot dipstick assay for SARS-CoV-2 Spike and Nucleocapsid antigens. The amount of 0.5 μg of each of S protein and N protein antigen (diluted in 10 mM sodium phosphate buffer, pH 7.4) was spotted on nitrocellulose dip sticks. The dip sticks were then incubated in 80 μL of sample buffer (1×PBS (pH 8), 5% BSA, 0.5% Casein, 0.2% Tween 20, 1% PEG 8000), 10 μL OD 5.5 conjugate (prepared as described above) and 0.135 μg (in 1 μL) of the respective antibodies for 20 minutes at room temperature. The results are shown in the photograph in
SARS-CoV-2 Spike or Nucleocapsid antibodies conjugated to the gold nanoparticles via the gold-fusion protein proteins were able to bind to the Spike or nucleocapsid antigen spotted onto the dipstick when wicked along the nitrocellulose membrane (Strips 3 and 4). More antibodies seemed to bind to the Nucleocapsid antigen compared to the Spike protein antigen. No signal was detected when only gold nanoparticles with gold-binding fusion conjugates were wicked along the membranes (Strips 1 and 2).
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) sequence coverage analysis:
Proteins are first digested to peptides by appropriate enzymes, such as Trypsin. Then, the peptide mixture is separated by liquid chromatography. Finally, the MS1 and MS2 spectrums of each peptide are detected by mass spectrometry.
Bioanalytical software matches the observed MS1 and MS2 spectrums to theoretical values to identify each peptide of the protein, and then calculates the peptide (or amino acid) coverage rate.
A 50 μL protein sample was diluted by 50 mM Tris-HCl to make a final concentration of 0.2 mg/mL. Then, 0.1M DTT was added at 1:20 DTT-to-protein volume ratio to reduce the disulfide bonds. After that, trypsin was added at 1:40 trypsin-to-protein mass ratio for 6 h digestion.
Finally, peptides were dried and re-diluted use 20 μL 0.1% FA-H2O for UPLC-MS analysis. UPLC Separation:
Column temperature 50° C., Flow rate 300 μL/min, Mobile Phase Solvent A: 0.1% FA-2% ACN in Water, Solvent B: 0.1% FA-90% ACN in Water
Electrospray voltage 3.5 kV, m/z scan range 200-2000 Ion transfer tube temperature , 333° C. , AGC 2e5, Resolution of MS120000, Collision energy 32 eV, Resolution of MS/MS 15000; Threshold ion count, 20000 ions/s.
BioPharma™ Finder™ 3.0 was used for LC-MS/MS data analysis. Results: The sequence coverage was 94.0% for the ISBP-free fusion protein (
Comparative binding analysis evaluating the direct binding of gold-fusion protein onto a gold sensor versus traditional EDC-NHS conjugation onto a gold sensor.
The immobilization of the gold-binding fusion protein which is Protein G (SEQ ID NO:19 with a linker [SEQ ID NO: 2] fused to gold binding protein SEQ ID NO: 4 (fusion known as “EMT-003”) and a reference sample onto gold sensor chips using direct immobilization (
As shown in Table 5 below, the gold-binding fusion protein showed a three-fold increase in Resonance Units (RU) during the immobilization phase by direct binding (2300 RU) compared to EDC-NHS process (750 RU). These results show that direct immobilization on gold is significantly more efficient than the immobilization using the EDC-NHS Process.
Evaluating the sensitivity and limit of detection (LoD) for the binding of SARS-CoV-2 Spike protein antigen and SARS-CoV-2 Nucleocapsid protein antigen to SARS-CoV-2 Spike and Nucleocapsid antibodies conjugated to gold-binding fusion protein on gold sensors prepared by direct immobilization or EDC-NHS conjugation:
First, gold sensors immobilized with gold-fusion protein by direct binding or EDC-NHS techniques according to Example 9, were conjugated with SARS-CoV-2 Spike or Nucleocapsid antibodies, and then SARS-CoV-2 Spike antigen or the negative control (SARS-CoV-2 Nucleocapsid antigen) following the method outlined in Example 3.
Surface plasmon resonance (SPR) was used to evaluate the sensitivity and LoD for the binding of SARS-CoV-2 Spike protein antigen and SARS-CoV-2 Nucleocapsid protein antigen to SARS-CoV-2 Spike or nucleocapsid antibodies conjugated to gold-fusion protein, which was immobilized on gold sensors by direct binding or EDC-NHS immobilization techniques from Example 9. As shown in
The detection of nucleocapsid antigen using the direct binding EMT-003 gold fusion protein-based SPR system in saliva (human, pooled) was then evaluated. As shown in FIG. 11A and 11B, recombinant nucleocapsid antigen binding was visible at all dilution. Detection was highest at 1:2 saliva in Running Buffer.
SARS-CoV-2 Spike Protein Detection by SPR: This example evaluated the performance of EMT003 coupled to an antibody for the selective detection of antigens under SPR. Specifically, EMT003 coupled to a SARS-CoV-2 anti-spike protein antibody was evaluated for the selective detection of spike protein. EMT003 was diluted to 10 μg/mL. Next, a clean gold coated sensor for SPR was loaded into the flow modules in the instrument. 500 μL of distilled water and 500 μL of PBS were flowed over the sensors briefly to establish the baseline signal. The fusion protein EMT003 was then flowed over the sensor for 10 minutes. Then 500 μL of PBS was flowed over the gold surface to removed poorly adsorbed EMT003 fusion protein. All measurements were performed at room temperature.
Two different types of antibodies were coupled to EMT003 over multiple SPR channels. First, 10 μg/mL of an anti-spike antibody was flowed over in channels B, C and D. As a negative control, 10 μg/mL of anti-TGFB was injected in channel A. Then, two wash steps with PBS and PBST were performed to remove excess of poorly absorbed antibody to EMT003. Finally, a blocking step with BSA was included to prevent potential non-specific binding to the sensor surface of spike protein during the titration step.
The titration with clinically relevant concentrations of SARS-CoV-2 spike protein consisted of four injections at gradually increasing concentration of: 10, 50, 100 and 200 ng/mL. The SPR real time bind profile is provided in
An additional titration of with high concentrations of SARS-CoV-2 spike protein consisted of five injections at gradually increasing concentration of: 300, 625, 1250, 2500 and 5000 ng/mL was also performed. This is indicated in
Conclusion: EMT003 coupled with anti-spike antibody was able to detect as low as 100 ng/mL of recombinant spike antigen. EMT003 coupled with anti-spike antibody can detect higher concentrations of recombinant spike protein in a linear and specific manner. The test is also specific, as EMT003 coupled with anti-TGFB did not detect spike protein as expected for the negative control.
Generation and purification of streptavidin fusion proteins in Escherichia coli:
The protein sequences for the fusion proteins, containing gold-binding peptides from Table 6 below, fused to a linker and streptavidin were converted to Streptavidin fusion proteins in an E. coli pET-30a (+) expression vector using the same cloning and purification strategy described in Example 1.
As shown in
Lateral flow assay application of streptavidin fusion proteins: Gold-binding streptavidin fusion proteins EMT027 and EMT028 were conjugated to gold nanoparticles according to the method outlined in Example 5. Both gold binding streptavidin fusion proteins bound successfully to gold nanoparticles across a range of pH.
Immobilization of biotinylated detection antibodies onto gold-binding streptavidin fusion protein coated gold nanoparticles and their antigen binding capacity was then tested using a lateral flow assay. In this assay, the antigen (rabbit IgG antibody) was directly dotted on the strip membrane. Biotinylated detection antibody (anti-rabbit IgG) was loaded onto streptavidin fusion proteins (EMT027 and EMT028) immobilized on gold nanoparticles, and then allowed to flow up the membrane. As shown in
The nucleocapsid antigen binding capacity of the EMT028-based gold nanoparticle conjugate was then tested in a ‘dotted’ sandwich lateral flow assay. In this assay, polyclonal anti-nucleocapsid antigen capture antibodies (chicken, top and rabbit, bottom) were dotted on the membrane. The EMT028-based gold nanoparticle conjugate was then mixed with nucleocapsid antigen and allowed to flow up the membrane. As shown in
The specificity of the EMT028-based gold nanoparticle conjugate system for nucleocapsid antigen was tested in a striped sandwich lateral flow assay. As shown in
The detection of nucleocapsid antigen at 1 ng/ml and 5 ng/ml in artificial saliva with mucin by EMT028 conjugate was also evaluated. In this assay, a sample volume of 60 uL was applied to each lateral flow strip. As shown in
Screening of nucleocapsid antibody using EMT028/biotin-nucleocapsid on SPR:
This study was performed to evaluate streptavidin fusion protein EMT028 coupled with SARS-CoV-2 biotinylated nucleocapsid protein for antibody detection as the analyte using SPR.
First, EMT028 was diluted to 10 μg/mL. Next, a clean gold coated sensor was loaded into the flow modules in the SPR instrument. 500 μL of distilled water and 500 μL of PBS were flowed over the sensors briefly to establish the baseline signal. The fusion protein EMT028 was then flowed over the sensor for 10 minutes. Then PBS and PBS-Tween (0.005%) was flowed over the gold surface to removed poorly adsorbed EMT028 fusion protein.
As a second layer in the system, a biotinylated nucleocapsid protein was coupled to EMT028. Then, one wash step with PBST was performed to remove excess of biotinylated protein. Finally, a blocking step with 1% BSA was included to prevent potential non-specific binding. 10 μl/mL of anti-nucleocapsid antibody MM08 was flowed in channel A, whereas an anti-spike antibody was injected in channel B (as a negative control). See
The interaction between anti-nucleocapsid MM08 antibody and biotinylated nucleocapsid protein showed a significant increase in the signal shift. This signal remained constant even after two PBST rinses suggesting a strong and stable binding. No shift in signal was observed when anti-spike was flowed over EMT028/biotin-nucleocapsid no major signal shift was observed for the interaction between anti-spike 298 and biotinylated nucleocapsid protein.
Conclusion: EMT028 coupled with biotinylated nucleocapsid protein was able to detect anti-nucleocapsid MM08 antibody at a concentration of 10 μg/mL, with no detection of binding to a non-nucleocapsid antibody, indicating a detection system that is both sensitive and specific.
Generation and purification of gold-binding and bispecific Immunoglobulin A and Bispecific Antibody fragments. Bispecific antibodies and antibody fusion fragments are made as known in the art. Specifically, the genes of different antibodies or antibody fragments are cloned and transfected into Expi-CHO cells (Thermofisher), then were purified by AKTA Explorer protein purification system.
A bispecific immunoglobulin A dimer is cloned, expressed and purified wherein one antibody monomer has high affinity for gold and the other antibody monomer of the fused immunoglobulin A dimer has a high affinity for SARS-CoV-2 Spike protein.
Surface plasmon resonance (SPR), an opto-electronic biosensing technique, is chosen to evaluate the binding kinetics of the bispecific immunoglobulin A fusion to a gold surface. First, the immobilization of the bispecific immunoglobulin A fusion and two controls (ISBP-free fusion protein and buffer only) is evaluated. Zero or minimal binding is observed for those controls. The bispecific immunoglobulin A fusion, however, shows a ten-fold increase in Resonance Units (RU) during the immobilization phase compared to the ISBP-free control version. After it is established that bispecific immunoglobulin A fusion is bound to the gold surface, a dilution series for the Spike antigen is performed spanning the following concentrations in two experiments: 1.5625 nM (×2), 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. The raw data is analyzed using BiacoreTM 8K Evaluation software version 1.1. It is shown that -CoV-2 Spike antibody binds to the bispecific immunoglobulin A fusion with a KD of between 1 to 2 E-10 M.
In another example, a bispecific antibody fragment fusion with a gold binding VH domain and a scFv specific to SARS-CoV-2 Spike protein is cloned, expressed and purified using various methods known in the art. In a specific example, the fusions will be cloned into a phagemid or other known cloning vector. The fusions, which comprise a 6× His-tag, and are to be cloned into an expression vector and transformed in the BL21 (DE3) competent cell line and expression system. The transformation is performed under such a condition that heat shock is performed in ice→42° C.×90 sec→in ice. 750 μL of LB medium is added to the BL21 solution transformed by heat shock, and the whole was cultured with shaking for 1 hour at 37° C. After that, centrifugation is performed at 6,000 rpm×5 min, and 650 μL of the culture supernatant is discarded. The remaining culture supernatant and a cell fraction as a precipitate is stirred and inoculated on an LB/amp. plate, and the whole is left standing at 37° C. overnight.
Once a clone is confirmed to have the intended fusion protein, a preculture solution with the clone is subcultured in 750 ML of a 2×YT medium, and the culture is further continued at 28° C. When OD600 exceeded 0.8, IPTG is added to have a final concentration of 1 mM, and culture is performed at 28° C. overnight.
The fusion protein is purified from an insoluble granule fraction through the following steps:
The culture solution is centrifuged at 6,000 rpm×30 min to obtain a precipitate as a bacterial fraction. The resultant is suspended in a Tris solution (20 mM Tris/500 mM NaCl) in ice. The resultant suspension is then homogenized with a French press to obtain a homogenized solution. Next, the homogenized solution is centrifuged at 12,000 rpm×15 min, and the supernatant is removed to obtain a precipitate as an insoluble granule fraction comprising the inclusion bodies.
The insoluble fraction is then immersed overnight in 10 mL of a 6 M guanidine hydrochloride/Tris solution. Next, the resultant is centrifuged at 12,000 rpm×10 min to obtain a supernatant as a solubilized solution.
A Ni column is used as a metal chelate column carrier. Column adjustment, sample loading, and a washing step are performed at room temperature (20° C.). Elution of a His tag-fused fusion protein as a target is performed in a 60 mM imidazole/Tris solution.
The sample comprising the fusion proteins is refolded using dialysis and is immersed in a 6 M guanidine hydrochloride/Tris solution and dialyzed for 6 hours while being gently stirred. The concentration of the guanidine hydrochloride solution of the external solution is slowly reduced over time in a stepwise manner into a PBS buffer wherein the fusion with a gold binding VH domain and a scFv specific to SARS-CoV-2 Spike protein is refolded appropriately.
Surface plasmon resonance (SPR), an opto-electronic biosensing technique, is chosen to evaluate the binding kinetics of a bispecific antibody fragment to a gold surface. First, the immobilization of the bispecific antibody fragment fusion and two controls (ISBP-free fusion protein and buffer only) is evaluated. Zero or minimal binding is observed for those controls. The bispecific antibody fragment fusion, however, shows a ten-fold increase in Resonance Units (RU) during the immobilization phase compared to the ISBP-free control version. After it is established that bispecific antibody fragment fusion is bound to the gold surface, a dilution series for the Spike antigen is performed spanning the following concentrations in two experiments: 1.5625 nM (×2), 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. . The raw data is analyzed using Biacore™ 8K Evaluation software version 1.1. It is shown that -CoV-2 Spike antibody binds to the bispecific antibody fragment fusion with a KD of between 1 to 2 E-10 M.
Binding analysis of synthetic binding proteins to silica, polystyrene, and cellulose fused to streptavidin and sensors using QCM-D:
The protein sequences for the fusion proteins, containing cellulose, polystyrene or silica binding peptides from Table 7 below, fused to a linker and streptavidin were converted to fusion proteins in an E. coli pET-30a (+) expression vector using the same cloning and purification strategy described in Example 1.
For analyte detection, the Table 7 fusion proteins were loaded on the respective silica, polystyrene, or cellulose sensors as the target surface as indicated in Table 7, using quartz crystal microbalance with dissipation monitoring (QCM-D). All Table 7 fusion proteins were diluted in in 1×PBS solution in Type 1 water to a concentration of 25 μg/ml.
BSA was diluted to 100 μg/mL using the same PBS solution. All biotinylated antibodies for binding to the streptavidin and the respective antigens for detection were diluted in 1×PBS solution in Type 1 water to a concentration of 25 μg/ml. This includes Troponin (antigen), anti-Troponin antibody, and biotinylated troponin antibody.
Each QCM sensor was primed with PBS for about 3 hrs; each sensor was then washed with new PBS for 5 min. Each fusion peptide diluted in PBS solution was loaded on the respective sensor with the indicated inorganic surface for 1 hr. After absorption of the fusion peptide to the surface, the sensor was washed with 30 min of PBS, followed by 30 min of BSA solution, followed by 30 min of PBS. A biotinylated troponin antibody was then loaded on to the surface for 40 min, followed by 30 min of PBS. Troponin antigen was then added for 15 min, followed by another 30 min wash of PBS.
Table 8 and 9 below summarizes the modeled mass and, thickness values for each step of these QCM sensor experiments. The sensorgrams are indicated in
Regardless, after fusion protein binding, there was minimal absorption of BSA blocking agent, but substantial absorption of the biotinylated troponin antibody indicating selective binding to streptavidin. Detection of binding to the intended antigen (troponin) is also detected in both.
Bispecific scFv antibodies:
In another example, a bispecific antibody fragment fusion with a gold binding VH domain and a scFv specific to troponin was cloned, expressed and purified using various methods known in the art. The scFv Troponin fusion (GL007) includes the sequence below in Table 10 and as diagramed in
Specifically,
This fusion was cloned into an expression vector and expression system well known in the art. The fusion protein is purified from an insoluble granule fraction through the following steps:
The culture solution was centrifuged at 6,000 rpm×30 min to obtain a precipitate as a bacterial fraction. The resultant was suspended in a Tris solution (20 mM Tris/500 mM NaCl) in ice. The resultant suspension was then homogenized with a French press to obtain a homogenized solution. Next, the homogenized solution was centrifuged at 12,000 rpm×15 min, and the supernatant was removed to obtain a precipitate as an insoluble granule fraction comprising the inclusion bodies.
The insoluble fraction was then immersed overnight in 10 mL of a 6 M guanidine hydrochloride/Tris solution. Next, the resultant wascentrifuged at 12,000 rpm×10 min to obtain a supernatant as a solubilized solution.
A Ni column was used as a metal chelate column carrier. Column adjustment, sample loading, and a washing step was performed at room temperature (20° C.). Elution of a His tag-fused fusion protein as a target was performed in a 60 mM imidazole/Tris solution.
The sample comprising the fusion proteins was refolded using dialysis and was immersed in a 6 M guanidine hydrochloride/Tris solution and dialyzed for 6 hours while being gently stirred. The concentration of the guanidine hydrochloride solution of the external solution was slowly reduced over time in a stepwise manner into a PBS buffer wherein the fusion with a gold binding VH domain and a scFv specific to Troponin was refolded appropriately.
The fusion protein and Troponin antigen was diluted in in 1×PBS solution in Type 1 water to a concentration of 25 μg/ml. BSA was diluted to 100 μg/mL using the same PBS solution.
The QCM sensor was then primed with PBS for about 1 hr and then was washed with new PBS for 5 min. The scFv Troponin fusion diluted in PBS solution was loaded on two Gold surface sensors for 1 hr. After absorption of the fusion peptide to the surface, the sensors were washed with 30 min of PBS, followed by 30 min of BSA solution, followed by 30 min of PBS. Troponin antigen or Spike Antigen control was then added to the respective sensor for 15 min, followed by another 30 min wash of PBS.
After adsorption of GL007 on gold sensor, negligible thickness changes during subsequent PBS rinsing step and BSA blocking step are detected. While the Troponin Antigen shows some initial adsorption to sensor, minimal final troponin antigen adsorption was observed after PBS rinsing.
Lateral flow assay streptavidin fusion proteins:
GL011 was produced by initially being cloned and amplified in the recombinant baculovirus Sf9 insect cell system. The gene to GL011 was inserted into plasmid DNA as known in the art using the QIAGEN miniprep DNA purification kit. Sf9 cells were also seeded in insect cell medium in a six-well tissue culture plate and allowed to attach.
For transfection 0.2 micrograms of DNA, 0.8 micrograms of baculovirus transfer vector DNA, 4 microliters of cellFectin reagent and 0.8 milliliters of FBS/antibiotics free medium was mixed and incubated at RT for 15 minutes. The medium from the cells was replaced with 2 milliliters of FBS/antibiotics free medium. The wash medium was removed and the transfection mix complex was overlayed onto the washed cells at 60 rpm, shaking for 4 hrs at 27 degrees Celsius. Once transfection of the recombinant baculovirus with GL011 gene, the baculovirus was amplified in T75 flasks with Sf9 cells per the SignalChem Pharmaceutical Sf9 amplification system.
To express the recombinants GL011 protein, 3×108 Sf9 cells in 300 ml of Excell-400 medium from JHR Biosciences were combined with about 5 MOI baculovirus in a spinner flask for shaking at 80 RPM for 72 hrs at 27 degrees Celsius. The Sf9 cells are then harvested by centrifugation of the medium and the removal of the supernatant. The pellet is the lysed and purified with the His-Tag on the GL011 protein by using the Talon Cobalt beads system.
Gold-binding streptavidin fusion protein GL011 was then conjugated to gold nanoparticles according to the method outlined in Example 5.
Immobilization of biotinylated detection antibodies onto gold-binding streptavidin fusion protein coated gold nanoparticles and their antigen binding capacity was then tested using a lateral flow assay. In this assay, the antigen, (SARS-CoV-2 Nucleocapsid antigen), was directly dotted on the strip membrane at various concentrations of antigen. Specifically, SARS-CoV-2 Nucleocapsid antigen was diluted in human pooled saliva at 100 ng/mL, 10 ng/mL, 2 ng/mL and then individually spotted on the lateral flow assay membrane.
Biotinylated detection antibody (SARS-CoV-2 nucleocapsid antibodies) was loaded onto streptavidin fusion protein GL011 immobilized on gold nanoparticles, and then allowed to flow up the membrane. As shown in
While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.
All publications, patents and patent applications, including any drawings and appendices therein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
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This application claims priority to U.S. Provisional Application No. 63/076,918, filed Sep. 10, 2020, and U.S. Provisional Application No. 63/163,695, filed Mar. 19, 2021, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051256 | 9/10/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63076918 | Sep 2020 | US | |
| 63163695 | Mar 2021 | US |
