Dual affinity probes and systems for analyte detection - Patents.com

Abstract

Described herein are dual affinity probes that include an organic or inorganic surface-binding peptide and a target-specific capture element that can bind to various targets, such as pathogens. Further described herein are uses of the dual affinity probes and kits for determining the presence and / or amount of targets in a sample. In certain embodiments, the dual affinity probes are specific for SARS-CoV-2 (spike or nucleocapsid) proteins and can be used to determine whether a subject is infected with SARS-CoV-2.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 318,365, filed March 9, 2022, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0002] The disclosed subject matter relates generally to the genetic assembly of inorganic and organic binding entities to function various biosensors for the detection of any pathogen of interest.

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing (GEML_002_01WO_SeqList_ST26.xml, size: 64,373 bytes, created on March 7, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] Pathogen detection for many applications relies primarily 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. Culture and ELISA are highly sensitive pathogen detection methods, but their main drawback is the turnaround time, which can take several days for culture to produce results. PCR is highly sensitive and faster than culture-based methods and immunoassays, but requires technical expertise and a multi-step process to first isolate DNA or RNA for analysis. Furthermore, PCR cannot distinguish between viable and non-viable pathogens.

[0005] Human coronaviruses are positive-sense, single-stranded RNA viruses. There are seven coronaviruses known to infect humans. Patients infected with these viruses develop respiratory symptoms of varying severity. HCoV-229E and HCoV-OC43 are well known and cause the common cold. Five other coronaviruses cause more severe respiratory tract infections that can be fatal. Since 2000, there have been three major global health outbreaks caused by coronaviruses (the SARS outbreak in 2003, the MERS outbreak in 2012, and most recently the COVID-19 outbreak in 2019).

[0006] Biosensors, analytical devices that combine a biological component with a physiochemical detector to detect chemicals, can be classified based on their capture element (enzyme-based, immunosensors using antibodies, DNA biosensors, etc.) or their transducer (thermal, piezoelectric biosensors, etc.). The best known biosensors are lateral flow-based pregnancy tests and electrochemical glucose biosensors.

[0007] The immobilization of capture elements or bioreceptors on surfaces is very important as it not only makes the biosensor functional but also determines its sensitivity. There are two groups of immobilization methods: irreversible and reversible. Irreversible immobilization includes covalent binding, cross-linking, and encapsulation, while reversible methods include random adsorption, bioaffinity (biotin / streptavidin and protein A / G), chelation / metal binding, and disulfide bonds (LIEBANA; DRAGO, 2016).

[0008] Antibodies are sensitive biomolecules that are often used in clinical applications of biosensors. The simplest method to prepare sensors using antibodies is random adsorption. However, random adsorption is associated with protein denaturation, very low stability, and random orientation, which affects the performance of biosensors. The most widely used method for immobilizing antibodies is by covalent bonding, which results in random orientation of the antibody because the amino / carboxyl groups used for covalent bonding are uniformly distributed on the antibody.

[0009] 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 specificity required for a variety of applications, including point-of-care environments. Summary of the Invention

[0010] The present disclosure provides dual affinity probes and related methods of use, for example, for determining the presence and / or amount or content of a target analyte in a sample. The dual affinity probes include (i) an inorganic surface binding element, and (ii) a capture element.

[0011] According to an embodiment of the present invention, a dual affinity immunoprobe for detecting an analyte, e.g., a pathogen, in a sample is provided, the immunoprobe comprising an inorganic surface-binding peptide and an analyte-specific capture element. In an embodiment, the analyte-specific capture element is an organic binding entity specific for the analyte, e.g., the 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 certain embodiments, the capture element specifically binds to the analyte, e.g., the pathogen.

[0012] In certain embodiments, the capture element is connected to the inorganic surface-binding peptide via a linker sequence. In yet other embodiments, the inorganic surface-binding peptide specifically binds to a biosensor material selected from the group consisting of gold, silica, silver, cellulose (e.g., nitrocellulose), plastic, polystyrene, and graphene.

[0013] In embodiments, the analyte-specific capture element specifically binds to an analyte. In embodiments, the analyte-specific capture element is a pathogen-specific capture element that specifically binds to a pathogen. In some embodiments, the pathogen is SARS-CoV-2.

[0014] In an embodiment of the invention, a dual affinity probe is provided in which the inorganic surface-binding peptide comprises a gold-, silver-, silica-, plastic-, cellulose-, polystyrene-, or graphene-binding peptide fused to protein G or streptavidin, and the capture element comprises an antibody that specifically binds to a target analyte.

[0015] In an embodiment of the invention, a dual affinity probe is provided in which the inorganic surface-binding peptide comprises a gold-, silver-, silica-, plastic-, cellulose-, polystyrene-, or graphene-binding peptide fused to protein G or streptavidin, and the capture element comprises a SARS-CoV-2 spike (S) antigen or a SARS-CoV-2 nucleocapsid (N) antigen-targeting antibody specific for S or N antigen.

[0016] In an embodiment, 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.

[0017] According to embodiments, a platform is provided for detecting the novel coronavirus SARS-CoV-2, in some embodiments via the SARS-CoV-2 spike (S) antigen, and in other embodiments via the SARS-CoV-2 nucleocapsid (N) antigen, using a gold-binding peptide fused to protein G and coupled to an S antigen-targeting antibody.

[0018] According to another embodiment, a platform is provided for detecting the novel coronavirus SARS-CoV-2 via the SARS-CoV-2 nucleocapsid (N) antigen using a silica-binding peptide fused to protein G and coupled to an N antigen-targeting antibody.

[0019] According to yet another embodiment, a platform is provided for detecting the novel coronavirus SARS-CoV-2 via the SARS-CoV-2 spike (S) antigen using a gold-binding peptide fused to protein G and coupled to an S and N antigen-targeting antibody.

[0020] According to another embodiment, a platform is provided for detecting the novel coronavirus SARS-CoV-2 via the SARS-CoV-2 nucleocapsid (N) antigen using a silica-binding peptide fused to protein G and coupled to an S and N antigen-targeting antibody.

[0021] According to embodiments, a platform is provided for detecting the novel coronavirus SARS-CoV-2, in some embodiments via the SARS-CoV-2 spike (S) antigen, and in other embodiments via the SARS-CoV-2 nucleocapsid (N) antigen, using gold-binding peptides fused to streptavidin and coupled to S and N antigen-targeting antibodies.

[0022] According to embodiments, a platform is provided for detecting the novel coronavirus SARS-CoV-2, in some embodiments via the SARS-CoV-2 spike (S) antigen, and in other embodiments via the SARS-CoV-2 nucleocapsid (N) antigen, using cellulose-binding peptides, silica-binding peptides fused to streptavidin, or polystyrene-binding peptides that are then fused to streptavidin and bound to S and N antigen-targeting antibodies.

[0023] In an embodiment, the platform detects analytes, such as pathogens, via a quartz crystal microbalance with dissipation monitoring (QCM-D). In another embodiment, the platform detects analytes, such as pathogens, via surface plasmon resonance (SPR). In yet another embodiment, the platform detects analytes, such as pathogens, via lateral flow.

[0024] In a specific embodiment, the present 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), which 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 linkers (LI), each LI can be independently 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 is represented by the following formula (I) or formula (II): SBM-LI-CE(Ia) or CE-LI-SBM(IIa) has.

[0025] The capture element CE may be an organic binding entity specific for the analyte, which 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 Fab fragment, or an antigen.

[0026] In another embodiment, the LI comprises one or more linkers, each linker being independently a single bond, e.g., an ionic or covalent or non-covalent bond, or 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, the LI comprises or is protein G from Streptococcus or streptavidin from Streptomyces.

[0027] In another embodiment, the SBM or ISBP specifically binds to a biosensor material selected from the group consisting of gold, silica, silver, cellulose, nitrocellulose, plastic, polystyrene, and graphene. In a further embodiment, the biosensor material is selected from the group consisting of gold, cellulose, silica, and polystyrene.

[0028] In more specific embodiments, the SBM or ISBP is selected from the group consisting of a binding peptide, a protein, an antibody with affinity for an 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 of the peptide sequences in Table 1 herein.

[0029] 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 H In another embodiment, the SBM or ISBP is an antibody. In a more specific embodiment, the SBM or ISBP is an antibody specific for binding to gold.

[0030] In specific embodiments, a dual affinity probe for detecting an analyte in a sample may comprise i) a surface binding moiety (SBM) fused or bound to a non-analyte capture element (NACE), or ii) a surface particle fused or bound to a NACE or capture element (CE).

[0031] In a specific embodiment, the surface particle is coupled to a surface binding peptide (SBP), the SBP is fused or coupled to a non-analyte capture element (NACE), the NACE is further fused or coupled to a CE, and the DAP has the following Formula I or Formula II: (Surface Particle)-SBP-NACE-CE (Formula Ic), or (Surface particles)-SBP-CE (Formula IIc).

[0032] In another embodiment of the dual affinity probe, the CE is an antibody, or an antigen-binding fragment thereof, optionally an scFv or Fab. In a specific embodiment, the CE is an antibody, or an antigen-binding fragment thereof, which 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 a SARS-CoV-2 spike (S) antigen or SARS-CoV-2 nucleocapsid (N) antigen-targeting antibody, or an antigen-binding fragment thereof, specific for S or N antigen.

[0033] 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 bound to a streptavidin linker. In another specific embodiment, the CE is an antigen that binds to an antibody (or antibodies) that is the analyte to be detected. In a specific embodiment, the antigen protein is SARS-CoV-2 spike and / or SARS-CoV-2 nucleocapsid protein. In another specific embodiment, the antigen binds to the antibody to detect it. In another embodiment, the antibody or antibodies are targeting antibodies specific for SARS-CoV-2 spike (S) antigen or SARS-CoV-2 nucleocapsid (N) antigen, or antigen-binding fragments thereof. In another embodiment of the dual affinity probe, the LI is a single bond, such as a covalent bond, or a peptide or amino acid linker. In specific embodiments, the amino acid linker is an inert linker, e.g., to allow space between the CE and the ISBP or to provide some rigidity or flexibility to the combination of the CE and the SBM or ISBP. In specific embodiments, the dual affinity probe is a single fusion protein. In another embodiment, the CE and the SBM or ISBP are independently antibodies, or antigen-binding fragments thereof, optionally single-chain variable fragments. In specific embodiments, the ISBP is a single-chain variable fragment. In more specific embodiments, the single-chain variable fragment is a V H 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 V HA gold-binding motif is a single-chain variable fragment, and the CE is a single-chain variable fragment specific for an antigen. In another embodiment, the CE and one or both of the SBM or ISBP are antibodies. 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 for 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.

[0034] The present invention also includes a composition comprising one or more dual affinity probes.In certain embodiments, the composition is a liquid composition, and the dual affinity probe is, for example, in a buffer solution.In other embodiments, the composition is a solid composition to which the one or more dual affinity probes are bound or immobilized.In another embodiment, the composition can be added to the surface of an assay, system, or kit.

[0035] The present invention may also include a dual affinity probe integrated into a particular system or diagnostic system, for example, a particular point-of-care diagnostic system. Any diagnostic system including a dual affinity probe may be used. For example, in a specific embodiment, the system includes an analysis performed on a quartz crystal microbalance, a surface plasmon resonance (SPR), and / or an analysis performed via lateral flow. In a specific embodiment, the system is used for the detection of an analyte, for example, a pathogen of known sequence, including a dual affinity probe. In a specific embodiment, the dual affinity probe may be any probe described herein. The system may include, for example, 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. Particular embodiments may include a system or diagnostic kit for detecting an analyte in a sample, the kit comprising an analyte composition comprising: a) a surface, b) a surface binding moiety (SBM) bound to at least a portion of the surface, c) a reagent for collecting the sample, and d) at least one set of dual affinity probes. In particular embodiments, the kit or system comprises: a) a surface, b) a surface binding moiety (SBM) bound to at least a portion of the surface, the SBM comprising a surface binding peptide (SBP) and / or optionally a non-analyte capture element (NACE), c) a reagent for collecting the sample, and d) an analyte composition comprising at least one set of dual affinity probes as described herein.

[0036] The invention also includes a method of detecting an analyte, e.g., a pathogen, using a dual affinity probe to analyze a medium for the analyte, e.g., a pathogen. In a specific embodiment, the dual affinity probe can be any dual affinity probe described herein. In another embodiment of the method, the analysis is performed using surface plasmon resonance (SPR), on a quartz crystal microbalance with dissipation monitoring (QCM-D), and / or via lateral flow.

[0037] In a specific embodiment of the method of the present invention, the method comprises determining the presence and / or quantifying an analyte, e.g., a pathogen, in a test sample, the method comprising: a) contacting the test sample with a dual affinity probe comprising an inorganic surface-binding polypeptide and an analyte-specific capture element under conditions and for a time sufficient to allow the analyte present in the test sample to bind to the analyte-specific capture element, thereby forming a complex comprising the analyte bound to the dual affinity probe; b) determining the presence or absence and / or amount of analyte present in the complex or complex; c) the presence of the analyte in the complex or complex indicates the presence of the analyte in the test sample, and the amount of analyte in the complex or complex indicates the amount of analyte present in the test sample, thereby determining the presence and / or quantifying the analyte in the test sample.

[0038] In a specific embodiment of the method, 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 fungus, a protozoan, an insect, 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 an antibody or an antigen-binding fragment thereof specific for a SARS-CoV-2 spike (S) antigen or a SARS-CoV-2 nucleocapsid (N) antigen.

[0039] In another embodiment of the method, the inorganic surface-binding polypeptide or surface-binding peptide comprises one or more gold-binding peptides, silver-binding peptides, silica-binding peptides, plastic-binding peptides, cellulose-binding peptides, or graphene-binding peptides. In another embodiment, the inorganic surface-binding polypeptide comprises a peptide selected from any of the peptide sequences in Table 1 or Table 1a herein. In another embodiment, the dual affinity probe is bound to a 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 method, the specific contacting and / or determining is performed using a quartz crystal microbalance, surface plasmon resonance (SPR), or via lateral flow. In specific embodiments, a method may include a method of determining the presence and / or quantifying an analyte in a sample from a subject by testing the sample with any one of the diagnostic kits or systems described herein, the method comprising: a) contacting the test sample with a reagent; b) applying the test sample together with the reagent to a surface, thereby causing the test sample to flow laterally over the surface, thereby contacting the DAP composition on the surface; c) allowing the analyte present in the test sample to directly bind to the CE, thereby forming a complex comprising the analyte bound to the DAP; d) allowing the analyte complexed with the DAP to further flow laterally over the surface, thereby complexing with a surface binding moiety (SBM); and e) determining the presence and / or amount of the analyte in the test sample by the presence of DAP complexed to the SBM.

[0040] Further features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0041] [Figure 1]A-C show the expression and purity of gold- and silica-bound ISBP on a Coomassie-stained SDS-PAGE gel. 2 μg of BSA was added to lane 1 as a loading control. A shows the ISBP-free fusion protein. B shows the gold-bound fusion protein. C shows the silica-bound fusion protein. [Diagram 2] Mass and thickness of the captured pathogen layer formed during capture of SARS-CoV-2 spike protein antigen by SARS-CoV-2 spike antibody using QCM-D on a gold sensor (left panel). The right panel shows the same experiment using SARS-CoV-2 nucleocapsid protein antigen and SARS-CoV-2 spike antibody. The left y-axis shows layer thickness (nm) and the right y-axis shows deposited mass (ng / cm2). The x-axis is time in seconds. [Diagram 3] 1 is an SPR sensorgram of immobilization of gold-bound fusion proteins onto a gold sensor surface. A non-ISBP-containing fusion protein and "buffer only" were run in parallel as controls. Gold-bound fusion proteins are shown in blue, non-ISBP-containing fusion proteins are shown in red, and buffer control is shown in green. The y-axis shows resonance units (RU) and the x-axis shows time in seconds. [Figure 4] Figure 1 shows the association and dissociation of different concentrations of SARS-CoV-2 spike protein antibodies (capture elements, anti-S antibodies) on a gold sensor coated with a gold-binding fusion protein. The y-axis shows the relative RU response and the x-axis shows time in seconds. [Diagram 5] 1 is a line graph showing the association and dissociation of various concentrations of spike protein antigen (S protein) with immobilized SARS-CoV-2 spike antibodies (anti-S protein antibodies). The y-axis shows relative RU response and the x-axis shows time in seconds. [Figure 6] Binding of different amounts of gold-conjugated fusion protein (rows 5-8) and ISBP-free fusion protein (rows 1-4) to 40 nm gold nanoparticles over a range of pH values ​​is shown. [Figure 7]Photograph of a capillary dot blot assay. SARS-CoV-2 spike protein antigen (strips 1 and 3) and SARS-CoV-2 nucleocapsid protein antigen (strips 2 and 4) were placed on nitrocellulose paper strips, which were then immersed in a solution containing gold nanoparticles conjugated to gold-conjugated fusion proteins and either SARS-CoV-2 spike protein antibody (strip 3), SARS-CoV-2 nucleocapsid protein antibody (strip 4), or no antibody (strips 1 and 2). [Figure 8] 1 is an SPR sensorgram of immobilization of a gold-bound fusion protein (sample) on a gold sensor surface by direct binding of the gold fusion protein to the gold sensor, where the y-axis indicates resonance units (RU) and the x-axis indicates time in minutes. [Figure 9] 1 is an SPR sensorgram of immobilization of a gold-binding fusion protein (EMT003) on a gold sensor surface by NHS-EDC mediated binding of the gold fusion protein to the gold sensor, where the y-axis indicates resonance units (RU) and the x-axis indicates time in minutes. [Figure 10] Graph showing binding of spike protein antigen (S) and nucleocapsid protein antigen (NC) at various concentrations (ug / mL) and limits of detection (LoD) with NHS-EDC immobilized EMT003 fusion protein with SARS-CoV-2 spike or nucleocapsid antibodies, respectively (left panel) compared to non-NHS immobilized EMT003 fusion protein (right panel). The y-axis shows the relative RU response and the x-axis shows the antigen concentration (μg / mL). [Figure 11] 1 shows the detection of nucleocapsid antigen in saliva (human pool) at various NC concentrations using a direct binding EMT003 gold fusion protein-based SPR system after diluting saliva at 1:2, 1:5, 1:10 and 1:20 dilutions in running buffer. A is an SPR sensorgram detecting binding in real time. B shows the RU response to dilution of NC in running buffer. [Figure 12A]Figures 12A and 12B show SPR sensorgrams using the EMT003-SARS-CoV-2 anti-spike combination to detect titers of SARS-CoV-2 spike protein in three different channels, and the EMT003-anti-TGFB control in the fourth channel. In different channels, Figure 12A detects SARS-CoV-2 spike protein at titers between 10 and 200 ng / mL, and Figure 12B detects SARS-CoV-2 spike protein at titers between 300 and 5,000 ng / mL. [Figure 12B] Figures 12A and 12B show SPR sensorgrams using the EMT003-SARS-CoV-2 anti-spike combination to detect titers of SARS-CoV-2 spike protein in three different channels, and the EMT003-anti-TGFB control in the fourth channel. In different channels, Figure 12A detects SARS-CoV-2 spike protein at titers between 10 and 200 ng / mL, and Figure 12B detects SARS-CoV-2 spike protein at titers between 300 and 5,000 ng / mL. [Figure 13] Expression and purity of gold-conjugated streptavidin fusion proteins on a Coomassie stained SDS-PAGE gel. 2 μg of BSA was added in lane 1 as a loading control. A shows the intact gold-conjugated streptavidin fusion protein EMT027, and B shows the intact gold-conjugated streptavidin fusion protein EMT028. [Figure 14] Photographs of lateral flow assays showing detection of antigen immobilized on a strip membrane by EMT027 and EMT028 based conjugates (i.e., gold nanoparticle-streptavidin fusion protein-biotin conjugate detection antibody complex) at different pH (8.2, 8.7, 9.0, and 9.2 for EMT027, and 6.5, 7.0, 7.4, and 7.8 for EMT028). [Figure 15]FIG. 13 is a photograph of a "dotted" sandwich lateral flow assay showing detection of dotted nucleocapsid antigen at different concentrations (0.0 μg / ml, 0.001 μg / ml, 0.01 μg / ml, and 0.1 μg / ml) using EMT028-based gold nanoparticle conjugates loaded with a biotin detection antibody (anti-nucleocapsid) using two different IgG or polyclonal capture antibodies. [Figure 16] Photograph of a striped sandwich lateral flow assay showing detection of nucleocapsid antigen but not spike antigen using EMT028-based gold nanoparticle conjugates coupled with nucleocapsid antibodies. From left to right: negative control, spike antigen at 1 μg / ml, nucleocapsid antigen at 1 μg / ml. [Figure 17] Photograph of a lateral flow assay showing detection of 1 ng / ml and 5 ng / ml nucleocapsid antigen in artificial saliva containing mucin by EMT028-based conjugates, in which a sample volume of 60 μL nucleocapsid antigen (1 ng / ml or 5 ng / ml) in artificial saliva was added to each lateral flow strip. [Figure 18] FIG. 13 is an SPR sensorgram screen of nucleocapsid antibodies using EMT028 bound to biotinylated nucleocapsid, thereby showing detection of the antibody in the screen. [Figure 19] FIG. 1 is a diagram of an exemplary embodiment of the EMT003, EMT027 / EMT028, and GL003 affinity probes. [Figure 20] FIG. 1 shows an exemplary embodiment of a universal dual affinity probe, including a bispecific tandem scFv format (left) and a bispecific immunoglobulin A format (right). [Figure 21] 1 shows Coomassie stained SDS-PAGE gels demonstrating the expression and purity of cellulose-bound streptavidin fusion proteins EMT032 and EMT033 (A-B), polystyrene-bound streptavidin fusion proteins GL008 and GL009 (C-D), and silica-bound streptavidin fusion protein EMT029 (E). [Figure 22A] 1 shows the change in absorption of the cellulose-bound streptavidin fusion protein EMT032 by the QCM-D sensor. [Figure 22B] 1 shows the change in absorbance of the cellulose-bound streptavidin fusion protein EMT033 by the QCM-D sensor. [Figure 22C] 1 shows the change in absorbance of the polystyrene-bound streptavidin fusion protein GL008 by the QCM-D sensor. [Figure 22D] 1 shows the change in absorbance of the polystyrene-bound streptavidin fusion protein GL009 by the QCM-D sensor. [Figure 22E] 1 shows the change in absorbance by the QCM-D sensor of the silica-bound streptavidin fusion protein EMT029. [Figure 23] FIG. 2 shows a diagram of scFv troponin fusion (GL007) containing the amino acid sequence (SEQ ID NO:29). [Figure 24] 1 shows a Coomassie stained SDS-PAGE gel showing the expression and purity of bispecific antibody GL007. [Diagram 25] The change in absorbance of GL007 by the QCM-D sensor at each sensor is shown following addition of troponin antigen (A) and spiked antigen as a control (B). [Figure 26] FIG. 1 shows the purity of GL011 His-tagged gold-conjugated streptavidin fusion protein on a Coomassie stained SDS-PAGE gel. [Figure 27] Figure 2 shows lateral flow assay detection of nucleocapsid antigen when diluted in pooled human saliva at 100 ng / mL, 10 ng / mL, and 2 ng / mL and detected by a biotinylated detection antibody (SARS-CoV-2 nucleocapsid antibody) when bound to the streptavidin fusion protein GL011 immobilized on gold nanoparticles. [Figure 28]FIG. 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow assay kit, which also illustrates a method for detecting an analyte in a sample by forming a detection complex comprising gold nanoparticles, a streptavidin fusion protein, and a biotinylated analyte-binding antibody. [Figure 29] FIG. 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow assay kit and method for detecting an analyte in a sample by having the analyte compete with a gold nanoparticle-bound antigen for binding to an anti-analyte antibody, where the formation of an antigen-bound gold nanoparticle-streptavidin fusion protein-biotinylated antibody complex is observed at the test line. [Figure 30A] FIG. 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow assay kit and method for detecting an analyte in a sample by using two compositions in separate lyophilized beads: a first composition comprising antigen-conjugated gold nanoparticles and a second composition having a biotinylated anti-analyte antibody that binds to both the antigen and the provided analyte. [Figure 30B] FIG. 1 is an exemplary embodiment of a diagnostic lateral flow assay kit and a method for detecting an analyte in a sample by using two compositions in separate lyophilized beads. FIG. 2 shows a method for providing a positive control by binding to a membrane-immobilized anti-IgY antibody and gold nanoparticles coated with IgY antibodies. [Figure 31A] 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow assay kit and a method for detecting an analyte in a sample by using two analyte compositions comprising two different sets of antibodies. A kit is shown with a first composition in lyophilized beads comprising gold nanoparticles coated with an antigen and a mouse monoclonal anti-analyte antibody. A second composition is shown in another lyophilized bead comprising gold nanoparticles coated with a biotinylated anti-mouse IgG antibody and a chicken IgY antibody. [Figure 31B]FIG. 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow assay kit and a method for detecting an analyte in a sample by using two analyte compositions comprising two different sets of antibodies. FIG. 2 shows how a positive control is provided on the control line by using a membrane-immobilized anti-chicken IgY antibody and gold nanoparticles coated with chicken IgY. [Figure 32A] Figures 32A-C are diagrams of an exemplary embodiment of a diagnostic lateral flow assay kit and method for detecting an analyte in a sample by providing two compositions having either a mouse anti-analyte monoclonal antibody or a gold nanoparticle-streptavidin fusion protein-biotinylated secondary anti-mouse IgG antibody complex (Figure 32A). Figures 32B and 32C show the formation of a sandwich complex including a rabbit IgG antibody on a test line, the analyte of interest, a mouse IgG antibody, a biotinylated secondary antibody, gold nanoparticles, and a streptavidin fusion protein. [Figure 32B] Figures 32A-C are diagrams of an exemplary embodiment of a diagnostic lateral flow assay kit and method for detecting an analyte in a sample by providing two compositions having either a mouse anti-analyte monoclonal antibody or a gold nanoparticle-streptavidin fusion protein-biotinylated secondary anti-mouse IgG antibody complex (Figure 32A). Figures 32B and 32C show the formation of a sandwich complex including a rabbit IgG antibody on a test line, the analyte of interest, a mouse IgG antibody, a biotinylated secondary antibody, gold nanoparticles, and a streptavidin fusion protein. [Figure 32C] Figures 32A-C are diagrams of an exemplary embodiment of a diagnostic lateral flow assay kit and method for detecting an analyte in a sample by providing two compositions having either a mouse anti-analyte monoclonal antibody or a gold nanoparticle-streptavidin fusion protein-biotinylated secondary anti-mouse IgG antibody complex (Figure 32A). Figures 32B and 32C show the formation of a sandwich complex including a rabbit IgG antibody on a test line, the analyte of interest, a mouse IgG antibody, a biotinylated secondary antibody, gold nanoparticles, and a streptavidin fusion protein. [Diagram 33] 1 is a diagram of an exemplary embodiment of an exemplary gold detection conjugate and a capture complex.A diagnostic lateral flow sandwich assay and method for detecting an antigen in a sample by formation of a sandwich detection complex is shown. [Diagram 34] FIG. 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow sandwich assay kit and method for detecting an analyte in a sample using a sandwich biotin-tagged antibody and antibody-conjugated gold nanoparticles. [Diagram 35] FIG. 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow sandwich assay kit and method for detecting an analyte in a sample using a sandwich His-tagged antibody and antibody-conjugated gold nanoparticles. [Diagram 36] 1 is a diagram of an exemplary embodiment of a diagnostic lateral flow sandwich assay kit and method for detecting an analyte in a sample using an anti-analyte antibody fused onto gold nanoparticles and another set of anti-analyte antibodies grown on different species. The two sets of secondary antibodies grown on different species are fused to a membrane and form complexes on a test line (analyte-bound complex) and a control line (analyte-unbound complex). [Figure 37] FIG. 37 is a diagram of an exemplary embodiment of a diagnostic lateral flow sandwich assay kit and a method for detecting an analyte in a sample by using the assay of FIG. 36 (except that the gold nanoparticles and dual affinity probes are encapsulated in lyophilized beads). [Figure 38] 1 includes a diagram of an exemplary embodiment of a directly modified antibody comprising a surface-binding moiety fused to the C-terminus of the Fc region of the antibody heavy chain. In certain embodiments, the surface-binding moiety is bound to the surface as a linear peptide, while in other embodiments, the surface-binding moiety is bound to the surface via its 3D polypeptide structure. [Figure 39] FIG. 1 shows the results of a wet assay for detecting nucleocapsid antigen at three concentrations when EMT033 cellulose-bound streptavidin fusion protein was striped onto nitrocellulose for immobilization of capture antibody. [Diagram 40] Showing the results of a wet assay to detect nucleocapsid antigen at three concentrations when EMT033 cellulose-bound streptavidin fusion protein was striped onto cellulose (Whatman 43 paper) for immobilization of capture antibody. [Diagram 41] EMT033 shows the results of a dry lateral flow assay for detection of whole SARS-CoV-2 virus when cellulose-bound streptavidin fusion protein was striped onto cellulose (Whatman 43 paper) for immobilization of capture antibodies. [Diagram 42] Shown are the results of a dry lateral flow assay for the detection of SARS-CoV-2 nucleocapsid antigen when the capture antibody was immobilized by binding to a cellulose-bound streptavidin fusion protein or physisorbed without the fusion protein. [Figure 43A] Figures 43A-C show the change in absorbance by the QCM-D sensor upon addition of various PDMS-bound fusion motifs, namely GL014 (Figure 43A), GL015 (Figure 43B), and GL016 (Figure 43C), as well as biotinylated SARS-CoV-2 nucleocapsid antibody and SARS-CoV-2 nucleocapsid antigen. [Figure 43B] Figures 43A-C show the change in absorbance by the QCM-D sensor upon addition of various PDMS-bound fusion motifs, namely GL014 (Figure 43A), GL015 (Figure 43B), and GL016 (Figure 43C), as well as biotinylated SARS-CoV-2 nucleocapsid antibody and SARS-CoV-2 nucleocapsid antigen. [Figure 43C] Figures 43A-C show the change in absorbance by the QCM-D sensor upon addition of various PDMS-bound fusion motifs, namely GL014 (Figure 43A), GL015 (Figure 43B), and GL016 (Figure 43C), as well as biotinylated SARS-CoV-2 nucleocapsid antibody and SARS-CoV-2 nucleocapsid antigen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The following terms are defined as follows:

[0043] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequence to specifically bind to an antigenic epitope. Thus, the term "antibody" includes antibodies and antigen-binding fragments thereof, where an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binds to a specific target antigen. Thus, the term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelid antibodies (or VHH), sdAB (nanobody), diabody, 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.

[0044] Single domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Like whole antibodies, they are able to selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, sdAbs are much smaller than typical complete antibodies (~150-160 kDa) which consist of two heavy and two light chains of protein, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from the light chain and one from the heavy chain). The first single domain antibodies were engineered from heavy chain antibodies found in camelids. These were the V H Also called H fragment.

[0045] "Antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an 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 an antibody results in two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, a name that reflects the ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0046] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent, such as an antibody, and can be used in an animal to generate antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., the capture element of a dual affinity probe) is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In specific embodiments, the antigen is not obtained in the sample for detection. In another embodiment, the antigen is a recombinant antigen or is not derived from the sample and is instead provided in a test system or diagnostic kit. In another embodiment, the antigen can be provided in a test system or diagnostic kit to be a competing protein or peptide for the analyte.

[0047] The term "analyte" as used herein refers to a protein, peptide, antibody, chemical, or target from a sample or the like that is to be identified or measured. Non-limiting examples of analytes include pathogens, proteins, peptides, antibodies, chemicals, hormones, environmental toxins, steroids, etc. In specific embodiments, the sample is derived from a subject, such as a human.

[0048] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that comprises at least one CDR of an immunoglobulin heavy and / or light chain or Nanobody® (Nab) that binds to an antigen of interest, e.g., a pathogen. In this regard, an antigen-binding fragment of an antibody described herein can comprise one, two, three, four, five, or all six CDRs of the VH and VL from an antibody that binds to one or more analytes, e.g., a pathogen.

[0049] Aptamers can be composed of proteins and / or nucleic acids. Peptide aptamers are artificial proteins that have been selected or engineered to bind to a specific target molecule. These proteins consist of one or more peptide loops of variable sequence displayed by a protein scaffold. They are usually isolated from combinatorial libraries and are often subsequently improved by directed mutation or multiple rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind to cellular protein targets and exert biological effects including interference with normal protein interactions of their target molecules with other proteins. Nucleic acid aptamers are nucleic acid species that exhibit binding affinity for a given target with selectivity and specificity comparable to antibodies. Nucleic acid aptamers can be composed of either RNA or DNA. Nucleic acid aptamers can be generated via in vitro selection methods such as SELEX (systematic evolution of ligands by exponential enrichment) to exhibit affinity for a wide range of targets, from small ligands such as heavy metal ions and small molecules to larger ligands such as proteins and cells. Aptamer-ligand interactions are mediated through non-covalent forces, electrostatic interactions, hydrophobic interactions, π-π orbital stacking, and hydrogen-bonding interactions. Aptamers can be engineered entirely in vitro, are easily generated by chemical synthesis, have desirable storage properties, and elicit little or no immunogenicity for therapeutic applications. X-aptamers are a new generation of aptamers designed to improve the binding and versatility of conventional DNA / RNA-based aptamers. X-aptamers are engineered using a combination of natural and chemically modified DNA or RNA nucleotides. Base modifications allow for the incorporation of a variety of functional groups / small molecules into X-aptamers, broadening their applications and increasing the likelihood of successful binding compared to standard aptamers. Phosphothioate backbone modifications at selected positions improve nuclease stability and binding affinity without sacrificing specificity. Optimers also include other forms such as split optimers and optimer ligands.

[0050] The term "linker sequence" is intended to mean a sequence that bridges a surface-binding entity, e.g., an inorganic surface-binding entity, with an organic binding entity, e.g., a capture element. As used herein, a linker sequence may comprise one or both of an active linker and / or an inactive linker. Thus, a linker sequence may comprise, for example, the amino acid sequence of protein G from Streptococcus or streptavidin from Streptomyces, or may simply 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 and biologically derived molecules.

[0051] The term "surface-binding motif", "surface-binding moiety", or SBM is intended to mean a molecule that has a specific and selective affinity for organic or inorganic materials, such as, for example, polydimethylsiloxane (PDMS), zinc oxide (ZnO), gold, quartz, silica, silicon, silver, cellulose, nitrocellulose, plastic, polystyrene, and graphene. The SBM may be a peptide or polypeptide and may include additional components, such as NACE, fused or attached to the SBM. The term "inorganic surface-binding peptide" or ISBP is intended to mean a sequence of amino acids that has a specific and selective affinity for inorganic materials, such as gold, silica, or graphene. Thus, the ISBP may include, for example, a short peptide, a protein, an antibody or antibody fragment with affinity for inorganic surfaces, a camelid antibody, a modified antibody, a single chain variable fragment, or a Fab fragment, and may include additional components, such as NACE, fused or attached to the ISBP. Thus, an ISBP may comprise, for example, a short peptide, a protein, an antibody or antibody fragment with affinity for inorganic surfaces, a camelid antibody, a modified antibody, a single chain variable fragment, or a Fab fragment, and may include additional components such as NACE fused or conjugated to the ISBP. The term "surface-binding peptide" or SBP is intended to mean a sequence of amino acids that has a specific and selective affinity for a material or surface such as polydimethylsiloxane (PDMS), zinc oxide (ZnO), gold, quartz, silica, silicon, silver, cellulose, nitrocellulose, plastic, polystyrene, and graphene.

[0052] The term "biosensor" is intended to mean a component or device that uses a biological component to convert the detection of an analyte, e.g., a pathogen, into a measurable signal. The term "biosensor material" is intended to mean a conversion of a biological or chemical reaction into a measurable signal that is proportional to the analyte, e.g., a pathogen, of interest. The signal generated can be in the form of, for example, a change in heat, light, pH, mass, or charge.

[0053] The term "capture element" is intended to include any moiety, e.g., an antigen, biotin, protein G from Streptococcus, or streptavidin from Streptomyces, or a single chain variable fragment, or an antibody or binding fragment thereof, optionally an antibody being a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment, e.g., SARS-CoV-2 spike and SARS-CoV-2 nucleocapsid targeting antibodies, capable of binding to an analyte or target to be detected and / or quantified. In a specific example, the analyte is derived from a sample for detection or quantification.

[0054] As used herein, the term "non-analyte capture element" or NACE is intended to include any moiety, such as an antigen, biotin, protein G from Streptococcus, or streptavidin from Streptomyces, or a single chain variable fragment, or an antibody or binding fragment thereof, optionally an antibody being a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment, that does not specifically bind to an analyte, but instead binds to a non-analyte moiety or target molecule. For example, the non-analyte capture element may specifically bind to another moiety of a dual affinity probe or another moiety on the surface of a system or diagnostic kit.

[0055] The term "covalent fusion" is intended to mean the joining of two or more genes encoding 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 the addition or deletion of signal sequences. The term "polypeptide" or "protein" or "peptide" means one or more amino acid chains, each chain comprising amino acids covalently linked by peptide bonds, and the polypeptide or protein or peptide can include multiple chains non-covalently and / or covalently linked together by peptide bonds, i.e., proteins produced by naturally occurring cells, particularly non-recombinant cells, or genetically engineered or recombinant cells, and can include molecules having the amino acid sequence of a native protein or molecules having deletions, additions, and / or substitutions of one or more amino acids of the native sequence. Thus, a "polypeptide" or "protein" can include one (referred to as a "monomer") or multiple (referred to as a "multimer") of amino acid chains.

[0056] The term "fusion protein" refers to a protein that is composed of at least two different amino acid sequences and is produced in an organism such as E. coli or insect cells of Spodoptera frugiperda. An inorganic surface-binding peptide expressed with protein A or G or a linker is an example of a fusion protein.

[0057] Examples of "pathogens" include SARS-CoV-2, influenza virus, 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 papillomavirus (HPV), herpes simplex virus (HSV), and human T-lymphocytes. The pathogenic agents that cause infection or disease in mammals include viruses, bacteria, and the like, such as any of those disclosed herein, including, but not limited to, human thyroid gland (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, smallpox, 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 indicative of the presence of a disease-causing organism or virus and / or biomarkers of a disease-causing organism or virus, such as, for example, the spike and nucleocapsid proteins of human coronaviruses, including SARS-CoV-2, influenza hemagglutinin, 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, smallpox, West Nile virus, Zika virus, and chikungunya virus antigens.

[0058] The term "specifically binds" means that a molecule reacts or associates with a particular target molecule, e.g., a pathogen, more frequently, more rapidly, for a longer duration, and / or with a higher affinity than it does with an alternative molecule, e.g., a pathogen. Reading this definition, it is understood that a molecule that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" does not necessarily require (although it can include) exclusive binding.

[0059] For antibodies, KD is the equilibrium dissociation constant, which is the calculated ratio of Koff / Kon between an antibody and its antigen. The binding constant (Kon) is used to characterize the rate at which an antibody binds to its target. The dissociation constant (Koff) is used to measure the rate at which an antibody dissociates from its target. KD and affinity are inversely correlated. High affinity interactions are characterized by a low KD, rapid recognition (high Kon), and strong stability of the complex formed (low Koff). In certain embodiments, a dual affinity probe, or its capture element, binds its target with at least 1×10 -2 or less, at least 1 × 10 -3 or less, at least 1 × 10 -4 or less, at least 1 × 10 -5 or less, at least 1 × 10 -6 or less, at least 1 × 10 -7 or less, at least 1 × 10 -8 or less, at least 1 × 10 -9 Less than or equal to at least 1 × 10 -10 or less, at least 1 × 10 -11 or less, or at least 1 × 10 -12 For the purposes of the present invention, the KD is determined from the binding curve using a Biacore2000 measurement device according to the analysis software provided with the device.

[0060] The features and advantages of the subject matter of the present invention will become more apparent in view of the following detailed description of selected embodiments, as illustrated in the accompanying drawings. As will be appreciated, the subject matter disclosed and claimed can be modified in various respects, all without departing from the scope of the claims. Accordingly, the drawings and description should be regarded as illustrative in nature, and not as restrictive, with the full scope of the subject matter being set forth in the claims.

[0061] In this disclosure, the word "comprising" is used in an open-ended sense to mean that the elements following the word are included, but not excluding elements not specifically mentioned.

[0062] In embodiments that include or may include a specified feature or variable or parameter, it will be understood that alternative embodiments can consist of or consist essentially of such feature or variable or parameter. Reference to an element with the indefinite article "a" does not exclude the presence of more than one of the element, unless the context clearly requires that there be one or only one of the element.

[0063] In this disclosure, when numerical ranges are recited by endpoints, they include all numbers within that range, including all whole numbers, all integers, and all fractions (e.g., 1 to 5 may include 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.). In this disclosure, the singular forms "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds.

[0064] In this disclosure, the term "or" is generally used in its inclusive sense, unless the content clearly dictates otherwise.

[0065] The present disclosure provides specific polypeptide sequences. Those skilled in the art will understand that these specific sequences may be modified and still achieve the same result, e.g., binding to another polypeptide, surface, or analyte. Thus, the present disclosure includes any variant of the disclosed polypeptides, and all of their uses. In certain embodiments, the polypeptide variants each contain one or more amino acid modifications compared to the disclosed sequences. Modifications include, but are not limited to, amino acid substitutions, insertions, and deletions. The variant polypeptides may contain at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the sequences disclosed herein. As used herein, the terms "identity" and "identical" refer to the percentage of perfect matching residues in an alignment of a "query" sequence with a "subject" sequence (e.g., an alignment generated by the BLAST algorithm, etc.) with respect to a polypeptide sequence. Identity is calculated over the entire length of the subject sequence, unless otherwise specified. Thus, a query sequence shares at least x% identity to a subject sequence if, when the query sequence is aligned with the subject sequence, at least x% (truncated) of the residues in the subject sequence align as perfect matches to the corresponding residues in the query sequence. If the subject sequence has variable positions (e.g., residues designated with an X), alignment to any residue in the query sequence counts as a match.

[0066] The present disclosure provides compositions and methods for detecting the presence and / or amount of an analyte in a test sample. The present disclosure may include elements described in PCT application PCT / CA2021 / 051256, which is incorporated herein by reference in its entirety.

[0067] Aspects of the present disclosure relate to dual affinity probes, or specifically dual affinity immunoprobes, that can be used to determine the presence or absence of an analyte in a test sample, the dual affinity probes comprising an inorganic surface-binding polypeptide and an analyte-specific capture element.

[0068] Dual affinity probes In a specific embodiment, the composition may include a dual affinity probe, which may be used to detect an analyte, such as an infectious agent or pathogen, in a sample, and the dual affinity probe includes a surface binding motif (SBM), such as an inorganic surface binding peptide (ISBP), and a capture element (CE). In another embodiment, the dual affinity probe may be a dual affinity immunoprobe, which means that the probe may be utilized with the use of an antibody or an antibody fragment. For example, the SBM and / or the CE may include an antibody or an antigen-binding fragment thereof.

[0069] In specific embodiments, the CE may be a molecularly imprinted polymer (MIP), an aptamer, a nanobody, or an engineered protein.

[0070] In certain embodiments of the dual affinity probe, the SBM or ISBP is a peptide. In certain embodiments, the SBM or ISBP is an antibody or an antigen-binding fragment thereof, such as an scFv. Various surface-binding peptides are known in the art, and exemplary surface-binding peptides are disclosed herein.

[0071] In certain 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, such as an scFv. Antibodies that specifically bind to various pathogens, including but not limited to those disclosed herein, are known in the art and can be readily produced.

[0072] The present disclosure contemplates various formats of dual affinity probes. In certain embodiments, a dual affinity probe comprises one or more polypeptides that bind both to a specific surface and to one or more specific target analytes. In other embodiments, a dual affinity probe comprises two or more polypeptides, including a first polypeptide that binds to a specific surface and also comprises an active linker that binds to a specific member of a binding pair, such as a class of molecules, e.g., antibodies or streptavidin / biotin, and a second polypeptide that comprises a target-specific capture element, the second polypeptide being linked by the active linker. For example, the second polypeptide may comprise an antibody or antigen-binding fragment thereof that specifically binds to a target analyte and / or may comprise a member of a binding pair to which the other member of the binding pair present on the first polypeptide binds. Thus, while certain dual affinity probes specifically bind to one or more target analytes, e.g., pathogens, other dual affinity probes may be adapted to identify any of a variety of different target analytes depending on the nature of the capture element, i.e., the target analyte that binds. Diagrams of various exemplary configurations of dual affinity probes are shown in Figures 19 and 20.

[0073] In certain embodiments, the SBM and CE are present in the same polypeptide and may be fused directly to each other or fused to each other via one or more linkers, e.g., an inert linker, e.g., a bond or a glycine-serine linker, or an IgA J chain or a llama IgG hinge region. In certain embodiments, the analyte-specific capture element specifically binds to the analyte of interest. In certain embodiments, the analyte-specific capture element is an antibody, or an antigen-binding fragment thereof, e.g., an scFv. In certain embodiments, the dual affinity probe is a single fusion protein. In another embodiment, the CE and ISBP are 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 V H In a specific embodiment, V H The binding motif is gold V HIn another embodiment, the CE is a single chain variable fragment from an antibody. In a specific embodiment, the ISBP and the CE are fused as a bispecific antibody fragment.

[0074] In certain embodiments, the SBM and the CE are present in different polypeptides. For example, in certain embodiments, the dual affinity probe comprises a first polypeptide comprising an SBM and an active linker, and a second polypeptide comprising a CE, and the active linker can be attached to the second polypeptide comprising an analyte-specific capture element. In certain embodiments, the active linker is directly attached to the analyte-specific capture element, e.g., the active linker can be protein A, protein G, or anti-IgG (e.g., goat anti-human IgG), and the analyte-specific capture element can be an antibody or an antigen-binding fragment thereof. In other embodiments, the analyte-specific capture element is fused to a non-specific binding element that is directly attached to the active linker, e.g., the non-specific capture element can be biotin and the active linker can be streptavidin, or vice versa. In certain embodiments, protein G is fused to the N-terminus or C-terminus of the SBM via an inert linker, e.g., a peptide linker. In certain embodiments, streptavidin is fused to the N-terminus or C-terminus of the SBM via an inert linker, e.g., a peptide linker. In addition to biotin and streptavidin, a variety of other binding pairs are known in the art and can alternatively be used.

[0075] In certain embodiments, the capture element (CE) is connected to the SBM via a linker sequence (LI), which can be a single bond or an amino acid sequence, and which is further connected to the SBM, such as an ISBP. In certain embodiments, the linker (LS) comprises one or more inactive linkers (PL) and / or one or more active linkers (AL). The dual affinity probe can have the following formula (I) or formula (II): SBM-LI-CE(I) or CE-LI-SBM(II).

[0076] In certain embodiments, a dual affinity probe comprises at least two polypeptides comprising a first polypeptide of formula (IIIa) or (IIIb) below, where PL is an inert linker, such as a single bond or an inert peptide linker, AL is an active linker that binds to a polypeptide of formula IV(a) or (IVb), the active linker binder (ALB) is a polypeptide sequence that is bound by AL, LI is an inert linker, such as a single bond or an inert peptide linker, and ALB and AL can be absent or present:

[0077] SBM-PL-AL(IIIa)

[0078] AL-PL-SBM(IIIb)

[0079] ALB-PL-CE(IVa)

[0080] CE-PL-ALB(IVb).

[0081] In specific embodiments, in formula (I) or formula (II), (IIIa), (IIIb), (IVa), (IVb), or any other formula described herein, each formula can begin at the N-terminus or C-terminus of a protein or peptide. For illustration, SBM-PL-AL (IIIa) of formula IIIA allows for SBM at the N-terminus (N-terminal orientation), and formula IIIb allows for SBM at the C-terminus (C-terminal orientation). Each of the specific formulas and embodiments described herein includes and allows for both N-terminal and C-terminal orientations.

[0082] In specific embodiments, the SBM or ISBP is connected to an inorganic surface, which may include an inorganic surface of a biosensor or other biosensor material. Inorganic surfaces or biosensor materials to which the SBM or ISBP may be connected may include, for example, polydimethylsiloxane (PDMS), zinc oxide (ZnO), gold, silica, silver, cellulose, plastic, polystyrene, and graphene. In specific embodiments, the biosensor material is selected from the group consisting of gold, cellulose, silica, and polystyrene.

[0083] Dual affinity probes may use such materials in various forms of biosensors or diagnostic platforms, for example biosensors or platforms may use technologies such as quartz crystal microbalance, surface plasmon resonance (SPR), or with lateral flow assays.

[0084] The dual affinity probe may incorporate any SBM or ISBP or LI or CE described herein in any combination.

[0085] In other embodiments, a dual affinity probe (DAP) may be used to detect an analyte in a sample and may include i) a surface binding moiety (SBM) fused or bound to a non-analyte capture element (NACE), or ii) a surface particle fused or bound to a NACE or capture element (CE).

[0086] In specific embodiments, the surface particle can be any surface including, for example, polydimethylsiloxane (PDMS), zinc oxide (ZnO), gold, quartz, silica, silicon, silver, cellulose, nitrocellulose, plastic, polystyrene, and graphene, hi specific embodiments, the surface particle is gold.

[0087] In another embodiment, the particles may be in the form of beads and may be lyophilized. In a specific embodiment, the DAP may include an SBM having a surface-binding peptide (SBP) that specifically binds to the surface particles. In a specific embodiment, the SBP is a gold-binding protein or peptide.

[0088] In another embodiment, the NACE is one or more selected from the group consisting of a linker (LI), streptavidin, protein G, biotin, an antigenic protein or peptide, or an antibody or binding fragment thereof, the antibody is a non-fragmented antibody, a modified antibody, a single chain variable fragment from an antibody, or a Fab fragment, and the CE is an antibody or binding fragment thereof selected from the group consisting of a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, a Fab fragment, and an antigenic protein or peptide.

[0089] In specific embodiments, the DAP can have a specific binding order, for example, a surface particle is bound to a surface binding peptide (SBP), the SBP is fused or bound to a non-analyte capture element (NACE), and the NACE is further fused or bound to a CE, and the DAP has the following formula Ic or formula IIc: (Surface Particle)-SBP-NACE-CE (Formula Ic), or (Surface particles)-SBP-CE (Formula IIc).

[0090] In another embodiment, the NACE is one or more selected from the group consisting of a linker (LI), streptavidin, protein G, biotin, an antigenic protein or peptide, or an antibody or binding fragment thereof, the antibody is a non-fragmented antibody, a modified antibody, a single chain variable fragment from an antibody, or a Fab fragment, and the CE is an antibody or binding fragment thereof selected from the group consisting of a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, a Fab fragment, and an antigenic protein or peptide.

[0091] In one embodiment, the SBP comprises a nitrocellulose-binding protein / peptide, a silicon-binding protein / peptide, a cellulose-binding motif, a polystyrene-binding motif, and / or a silica-binding motif, hi another embodiment, the SBP comprises a sequence in Table 1 herein.

[0092] In a specific embodiment, the NACE comprises one or more selected from the group consisting of a linker, streptavidin, protein G, biotin, a biotinylated protein, an antigenic protein or peptide, and an antibody or binding fragment thereof, optionally the antibody is a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment. In a specific embodiment, the NACE comprises an antibody or binding fragment thereof, optionally the non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment, which specifically binds to IgG, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgD, and IgE, but preferably IgG. In another embodiment, the NACE comprises protein G and / or streptavidin. In another embodiment, the NACE comprises a binding tag, such as a His tag or a biotin tag. In another embodiment, the NACE comprises a cleavage site, such as a TEV cleavage site, or similar such sequences are known in the art.

[0093] In another embodiment, the CE is an antibody or binding fragment thereof, optionally the antibody or binding fragment thereof is selected from the group consisting of a non-fragmented antibody, a camelid antibody, a single chain variable fragment from an antibody, and a Fab fragment, or an antigenic protein or peptide. In another embodiment, the CE is a non-fragmented antibody. In another specific embodiment, the antibody comprises a tag sequence, optionally a biotin tag or a his tag.

[0094] In specific embodiments, the surface particle can be any surface including, for example, polydimethylsiloxane (PDMS), zinc oxide (ZnO), gold, quartz, silica, silicon, silver, cellulose, nitrocellulose, plastic, polystyrene, and graphene, hi specific embodiments, the surface particle is gold.

[0095] In another embodiment, the particles can be in the form of beads, such as analyte beads for specific binding to an analyte. In a specific embodiment, the analyte beads are freeze-dried. In another specific embodiment, the freeze-dried surface particles or analyte beads are in the size range of about 10 nm to about 80 nm. In another specific embodiment, the freeze-dried surface particles or analyte beads are in the size range of about 15 nm to about 60 nm. In another specific embodiment, the freeze-dried surface particles or analyte beads are in the size range of about 30 nm to about 50 nm.

[0096] In a specific embodiment, the dual affinity probes can be utilized for binding to a gold surface. In a specific embodiment, the gold surface can be in the form of particles, such as nanoparticles, and / or beads. In another embodiment, the gold surface can be bound to gold surface peptides / proteins.

[0097] In specific embodiments, the probe may comprise i) a surface binding moiety (SBM), which is a gold binding protein (GBP) fused or conjugated to a non-analyte capture element (NACE), or ii) a surface binding moiety (SBM), which is a gold binding protein (GBP) fused or conjugated to a capture element (CE).

[0098] In another specific embodiment, the gold particles of the probe are conjugated to a gold binding protein (GBP), which is fused or conjugated to a CE (Formula IIIc) or a NACE, which is further fused or conjugated to a CE (Formula IVc) shown below: (gold particle)-GBP-CE (Formula IIIc), or (gold particles)-GBP-NACE-CE (Formula IVc).

[0099] In another embodiment, the NACE comprises one or more selected from the group consisting of a linker, streptavidin, protein G, biotin, a biotinylated protein, an antigenic protein or peptide, and an antibody or binding fragment thereof, optionally wherein the antibody is a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment. In another embodiment, the NACE comprises an antibody or binding fragment thereof that specifically binds to IgG, optionally a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment.

[0100] In another embodiment, the CE is an antibody or binding fragment thereof, optionally the antibody or binding fragment thereof is selected from the group consisting of a non-fragmented antibody, a camelid antibody, a single chain variable fragment from an antibody, and a Fab fragment, or an antigenic protein or peptide. In a specific embodiment, the CE is a non-fragmented antibody. In another specific embodiment, the CE comprises a tag sequence, such as a biotin tag or a his tag.

[0101] In a specific embodiment, the fusion protein may comprise a sequence of Table 1, Table 1a, Table 6, Table 7, Table 10, and Table 12. In a specific embodiment, the fusion protein may comprise an amino acid sequence of Table 1a. In a specific embodiment, the fusion protein may consist of a sequence of Table 1, Table 1a, Table 6, Table 7, Table 10, and Table 12. In a specific embodiment, the fusion protein may consist of a sequence of Table 1a. In another specific embodiment, the fusion protein may comprise a sequence of Table 1, Table 1a, Table 6, Table 7, Table 10, and Table 12 with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In a specific embodiment, the fusion protein may consist of a sequence of Table 1a with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0102] In specific embodiments, the GBP may include sequences such as EMT014, EMT015, EMT016, EMT017, EMT018, and EMT019.

[0103] In a specific embodiment, the GBP is fused or conjugated to an additional NACE compound selected from the group consisting of protein G, streptavidin, biotin, and a biotinylated protein. In another embodiment, the NACE comprises protein G, which is fused to a gold-binding protein. In another specific embodiment, the NACE comprises streptavidin, which is conjugated to the GBP.

[0104] In specific embodiments, the GBP may comprise a sequence of Table 1 or Table 1a, either separately or as a fusion protein, e.g., EMT014, EMT015, EMT016, EMT017, EMT018, EMT019, EMT020, EMT021, EMT022, EMT023, EMT024, EMT003, EMT027, EMT028, and GL011.

[0105] In a specific embodiment, the GBP-NACE is a fusion protein selected from GL011, GL017, a GBP-protein G fusion (EMT03), and a GBP streptavidin fusion EMT027, or EMT028.

[0106] In another embodiment, the NACE further comprises an antibody or a Fab fragment specific for binding to IgG, or a biotinylated antibody.

[0107] In another embodiment, the gold particles can be in the form of beads, such as analyte beads for specific binding to an analyte. In a specific embodiment, the analyte beads are freeze-dried. In another specific embodiment, the freeze-dried surface particles or analyte beads are in the size range of about 10 nm to about 80 nm. In another specific embodiment, the freeze-dried gold surface particles or analyte beads are in the size range of about 15 nm to about 60 nm. In another specific embodiment, the freeze-dried gold surface particles or analyte beads are in the size range of about 30 nm to about 50 nm.

[0108] Capture element (CE) of dual affinity probe The capture element (CE) of the present invention may comprise any organic binding entity that binds to a particular analyte of interest. In certain 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, such as 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 can be readily generated. In specific embodiments, the capture element is a fragment of an antibody, such as a single chain variable fragment, or a Fab fragment.

[0109] 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 capture element is an aptamer or ligand. In certain embodiments, the capture element is an antibody, e.g., an scFv or nanobody. In certain embodiments, the methods disclosed herein can 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 utilized to test a sample of a subject to determine whether the subject has antibodies against a particular pathogen or infectious agent, more specifically, a particular antigen or epitope thereof that identifies the pathogen. Thus, in specific embodiments, the capture element comprises at least a portion of an antigen, or an epitope thereof, bound by one or more antibodies that specifically bind to the pathogen. In certain embodiments, the antigen may be any agent that can induce, for example, an immune response in a mammal that results in the production of antibodies that bind to the antigen.

[0110] The capture element may be specific for any analyte or pathogen of interest, for example, the capture element may be specific for an antigen, protein, peptide, or other organic moiety that identifies a subject as positive for or capable of infection with a particular pathogen. In a specific embodiment, the capture element is specific for an antigen to 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, 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 antibody may be a specific antibody listed in Table 2 herein.

[0111] Other pathogens for which the capture element may be specific include, by way of example and without limitation, coronavirus species, e.g., SARS and MERS, influenza species, respiratory syncytial virus species, adenovirus species, parainfluenza species, Filoviridae, e.g., Ebola and Marburg, Hantavirus species, Arenaviridae, e.g., Lassa, Bunyaviridae, e.g., Rift Valley and Crimean-Congo, and Paramyxoviridae, e.g., Hendra and Nipah. In some embodiments, the pathogen includes a prion. In some embodiments, the pathogen includes gram-negative and gram-positive bacteria. Other pathogens may include, for example, infectious diseases. The capture element may be specific for analytes or antigens in infectious diseases, e.g., Hepatitis B and C, HIV, syphilis, chlamydia, and gonorrhea.

[0112] In another embodiment, the capture element is an antigen and is specific to a 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 complete sequence of the spike protein or any variant thereof.

[0113] In another specific embodiment, the capture element (CE) is an antigen 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 bound to a streptavidin linker. In another specific embodiment, the CE is an antigen that binds to an antibody (or antibodies) that is the analyte to be detected. In a specific embodiment, the antigen protein is SARS-CoV-2 spike and / or SARS-CoV-2 nucleocapsid protein. In another specific embodiment, the antigen binds to the antibody and is detected. In another embodiment, the antibody or antibodies are targeting antibodies specific for SARS-CoV-2 spike (S) antigen or SARS-CoV-2 nucleocapsid (N) antigen, or antigen-binding fragments thereof.

[0114] In specific embodiments, the capture element may be linked to a linker (LI) or ISBP to ensure effective binding to the analyte of interest. For example, the spike protein of SARS-CoV-2 may be linked to a linker (LI) or ISBP to ensure that the correct portion of the protein or epitope is exposed to the analyte, in this case, antibodies that may be specific to various portions of the spike protein. Methods for binding capture elements or specific amino acid sequences to other amino acid sequences are known in the art and may be utilized in certain inventions described herein. For example, in another embodiment, the capture element may be tagged or modified for specific binding to a linker or direct binding to an ISBP. For example, the capture element may be biotinylated for only binding to a streptavidin linker, such as streptavidin from Streptomyces. In another embodiment, the capture element may be an antibody specific for IgG and protein G from Streptococcus, or an element modified to bind more efficiently to a linker, such as protein G.

[0115] Non-analyte capture elements of dual affinity probes The non-analyte capture element (NACE) may comprise any binding moiety, such as an antigen, biotin, protein G from Streptococcus, or streptavidin from Streptomyces, or a single chain variable fragment, or an antibody or binding fragment thereof, optionally where the antibody is a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment, but does not specifically bind to the analyte, but instead binds to a non-analyte moiety or target molecule.

[0116] The non-analyte capture element may specifically bind to another moiety of the dual affinity probe or to another moiety on the surface of the system or diagnostic kit.

[0117] In one embodiment, the NACE comprises one or more selected from the group consisting of a linker, streptavidin, protein G, biotin, a biotinylated protein, an antigenic protein or peptide, and an antibody or binding fragment thereof, optionally the antibody is a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment. In a specific embodiment, the NACE comprises an antibody or binding fragment thereof, optionally a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment, which specifically binds to IgG, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgD, and IgE, but preferably IgG. In another embodiment, the NACE comprises protein G and / or streptavidin. In another embodiment, the NACE comprises a binding tag, such as a His tag or a biotin tag.

[0118] In a specific embodiment, the NACE is attached or fused to any other element of the dual affinity probe and / or to the surface-binding domain. For example, the NACE can be attached or fused directly to a surface, a surface bead, a surface-binding protein / peptide, and / or a capture element.

[0119] Linker (LI) of dual affinity probes The dual affinity probes of the invention may include a linker, which may comprise any suitable amino acid sequence necessary to control steric hindrance and / or chemical interactions with the sensor components (organic or inorganic materials, peptides and proteins, cross-linking reagents, etc.).

[0120] The linker sequence of the dual affinity probe of the present invention may include one or more inert linkers and / or active linkers. In certain embodiments, the dual affinity probe includes an inert linker fused to an active linker, for example, to link an SBM or ISBP, or a NACE to the active linker. As used herein, an inert linker does not specifically bind to a capture element or other polypeptide, and typically resides between two polypeptide sequences to control steric hindrance, for example, to preserve the activity of the two linked polypeptides. In certain embodiments, the inert linker may be a single bond or an amino acid sequence that links an SBM, a NACE, or an ISBP to a CE (or a polypeptide that includes a CE). An inert linker may also reside between the CE and a member of a binding pair to which the CE is fused. The linkage may be a covalent bond, an ionic bond, a non-covalent bond, for example, using a high affinity molecule.

[0121] As used herein, an active linker may be fused to an SBM or ISBP and may be present to specifically bind to a CE or a polypeptide comprising a CE (e.g., a member of a binding pair present in a polypeptide comprising a CE) and operably link the SBM or ISBP to the CE. In certain embodiments, the active linker binds to an antibody or antigen-binding fragment thereof (e.g., a human antibody or fragment thereof). In certain embodiments, the active linker is a member of a binding pair such as streptavidin / biotin. The linkage may be covalent, ionic, or non-covalent, e.g., using a high affinity molecule.

[0122] In alternative embodiments, the linker sequence may comprise other amino acid sequences, such as an inert linker, a linear tandem repeat polypeptide, a linear non-repetitive polypeptide, or a linker that allows for additional flexibility or rigidity to the SBM, ISBP, or CE.

[0123] In specific embodiments, the high affinity molecule (i.e., AL) in the linker can be an amino acid sequence that includes protein G from Streptococcus, or an amino acid sequence that includes streptavidin from Streptomyces. In another embodiment, the linker can include an additional AL that directly and covalently binds to the SBM, ISBP, but with high affinity to IgG or biotin incorporated in the capture element.

[0124] In specific embodiments, the inert linker may comprise a glycine-serine linker, for example, the following amino acid sequence: GGGGSGGGGSGGGGSASGGG [SEQ ID NO: 1]

[0125] In another specific embodiment, the linker may have the following amino acid sequence: TPTPTTPTPTPTTPTPTPST [SEQ ID NO:61]

[0126] In another embodiment, the inert linker of SEQ ID NO:1 or SEQ ID NO:61 can be further incorporated or fused to a linker, e.g., another amino acid sequence of AL, e.g., a high affinity protein such as streptavidin or protein G. In a specific embodiment, SEQ ID NO:1 is fused directly to protein G to form the following sequence [SEQ ID NO:2]. MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNG VDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSASGGG

[0127] In this example, the inert linker SEQ ID NO:1 is on the C-terminus of the AL and directly links it to the SBM or ISBP, and the amino acid sequence of protein G binds with high affinity to the capture element, which may be any IgG antibody or a suitable fragment of an IgG antibody.

[0128] In another specific embodiment, an inert linker, such as SEQ ID NO: 1, can be fused to streptavidin (AL) at the linker. In a specific embodiment, the inert linker SEQ ID NO: 1 is on the C-terminus of the AL and directly links to the SBM or ISBP, and the streptavidin amino acid sequence binds with high affinity to biotinylated capture elements.

[0129] In a specific embodiment, SEQ ID NO:1 is fused directly to streptavidin to form the following sequence [SEQ ID NO:21]. MDPSKDSKAQVSAAEAAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQGGGGSGGGGSGGGGSASGGG

[0130] In this example, the inert linker SEQ ID NO:1 is on the C-terminus of streptavidin AL and directly links the SBM or ISBP, and the streptavidin amino acid sequence binds with high affinity to a capture element (or polypeptide comprising a CE), which may be a biotinylated protein including an antibody or antibody fragment.

[0131] In a specific embodiment, the ISBP fusion to the linker can be an amino acid sequence or peptide that binds to gold, silicon, cellulose, polystyrene, or silica. In another specific embodiment, the ISBP can be or include any one of SEQ ID NOs: 3-19 or 25.

[0132] In another embodiment, the linker sequence does not include an inactive linker. For example, the linker AL can be specific only to the amino acid sequence of protein G or the streptavidin amino acid sequence. In a specific embodiment, the linker (AL) can include the following sequence of protein G [SEQ ID NO: 19]. MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE

[0133] In a specific embodiment, the linker (AL) is SEQ ID NO:19.

[0134] In another embodiment, the linker (AL) may comprise the following sequence of streptavidin [SEQ ID NO: 22]: MDPSKDSKAQVSAAEAAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ

[0135] In specific embodiments, the streptavidin sequence may not be directly linked at the N-terminus of any peptide or protein and / or may not be directly linked to an N-terminal methionine residue. In specific embodiments, the streptavidin sequence may be as follows:

[0136] [SEQ ID NO: 60] below: DPSKDSKAQVSAAEAAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ

[0137] In another specific embodiment, the linker sequences may be their own fusion proteins or may incorporate other elements of the invention, such as SBM or ISBP and / or CE, to form fusion proteins. Fusion proteins, including their design, gene synthesis, cloning, expression, and purification, are known in the art and may be incorporated to form any fusion thereof. For example, the linkers of the invention may incorporate such sequences with tags or cleavage sites for protein purification, such as His tags or other protein tags known in the art. In another embodiment, the peptide or protein may include a protease or cleavage site. In a specific embodiment, the cleavage site may be a TEV cleavage site. The examples of this application provide examples of specific fusion proteins, but the invention herein is not limited thereto.

[0138] In another specific embodiment, the LI linker can simply be a single bond, such as a covalent bond. Thus, in such an example, the SBM or ISBP and the CE are directly bonded to each other, without any additional amino acids or atoms representing the linker.

[0139] Surface-binding portion of dual affinity probe The dual affinity probe of the present invention may comprise a surface-binding moiety (SBM) that binds to a selected organic or inorganic surface. For example, the SBM specifically binds to a biosensor material selected from the group consisting of gold, silica, silver, cellulose, e.g., nitrocellulose, plastic, polystyrene, and graphene. In certain embodiments, the SBM is an organic or inorganic surface-binding polypeptide (ISBP). As used herein, the ISBP may bind to an organic or inorganic surface. In another example, the ISBP may specifically bind to a biosensor material selected from the group consisting of gold, cellulose, silica, and polystyrene.

[0140] In specific embodiments, the SBM or ISBP may comprise an amino acid sequence and may be selected from the group consisting of a binding peptide, a protein, an antibody having affinity for an inorganic surface, or an antigen-binding fragment thereof, e.g., a single chain variable fragment (scFv). In certain embodiments, the inorganic surface-binding polypeptide is a peptide. In certain 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 exemplary surface-binding peptides are disclosed herein.

[0141] In specific embodiments, the ISBP comprises a peptide specific for binding to gold, cellulose, silicon, or polystyrene, hi another embodiment, the ISBP comprises a peptide of Table 1 provided herein.

[0142] In another embodiment, the ISBP comprises an antibody or a fragment of an antibody. H or V LIn a specific embodiment, the ISBP is a gold V binding motif. H or V L The ISBP is a binding motif. In a specific embodiment, the antibody or antibody fragment can be specific for binding to gold. In a specific embodiment, the ISBP can be the gold-binding protein of U.S. Pat. No. 7,807,391, Shiotsuda et al., which is incorporated herein by reference in its entirety.

[0143] ISBP-LI-CE (Ia) or (IIa) dual affinity probes 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).

[0144] In a specific embodiment, the capture element CE is an organic binding entity specific for a pathogen. The capture element is selected from a single chain variable fragment, a Fab fragment, an antibody, or an antigen, and the LI is a linker sequence comprising one or more inactive linkers and / or active linkers. In certain embodiments, one or more of the linkers present in the 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 specifically binds to a biosensor material selected from the group consisting of gold, silica, silver, cellulose, plastic, polystyrene, and graphene.

[0145] In a specific embodiment, LI is a single bond, thereby allowing ISBP to bind directly to CE.

[0146] In this configuration, the dual affinity probe may comprise an inorganic surface-binding polypeptide and an analyte-specific capture element within the same polypeptide, which may be fused directly to each other or fused to each other via one or more linkers, e.g., inert polypeptide linkers. In certain embodiments, the analyte-specific capture element specifically binds to the analyte of interest. In certain embodiments, the analyte-specific capture element is an antibody, or an antigen-binding fragment thereof, e.g., an scFv.

[0147] In a specific embodiment, the dual affinity probe is a single fusion protein. In another embodiment, the CE and the ISBP are 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 V H In a specific embodiment, V H The binding motif is gold V H In another embodiment, the CE is a single chain variable fragment from an antibody. In a specific embodiment, the ISBP and the CE are fused as a bispecific antibody fragment.

[0148] In certain combinations, ISBP is H A single chain variable fragment that is a gold-binding motif, and CE is a single chain variable fragment specific for an antigen.

[0149] In another particular 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. In this case, the CE and ISBP are, or independently, V L Fragment, V H fragment and / or scFv fragment.

[0150] In another specific embodiment, the ISBP is specific for gold, silica, silver, cellulose, plastic, polystyrene, and graphene, hi a specific embodiment, the ISBP is specific for gold.

[0151] In another specific embodiment, the CE is specific for an antigen against SARS-CoV-2.

[0152] In a specific embodiment, the CE is specific for the SARS-CoV-2 spike (S) antigen or the 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 the SARS-CoV-2 spike (S) antigen or the SARS-CoV-2 nucleocapsid (N) antigen.

[0153] In another particular combination, the CE and ISBP are each an antibody with a linker between them. 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. In this case, the CE and ISBP are, or independently, V L Fragment, V H fragment and / or scFv fragment.

[0154] 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 bound to a streptavidin linker. In another specific embodiment, the CE is an antigen that binds to an antibody (or antibodies) that is the analyte to be detected. In a specific embodiment, the antigen protein is SARS-CoV-2 spike and / or SARS-CoV-2 nucleocapsid protein. In another specific embodiment, the antigen binds to the antibody and is detected. In another embodiment, the antibody or antibodies are targeting antibodies specific for SARS-CoV-2 spike (S) antigen or SARS-CoV-2 nucleocapsid (N) antigen, or antigen-binding fragments thereof.

[0155] ISBP-LI-CE (IIIc, IIId, IVc, IVd) dual affinity probes The dual affinity probes of the invention may comprise one or more polypeptides having formula (IIIc) or (IIId) and one or more polypeptides having formula (IVc) or (IVd).

[0156] IRISB-PL-AL(IIIc)

[0157] AL-PL-ISBP(IIId)

[0158] ALB-PL-CE(IVc)

[0159] CE-PL-ALB(IVd)

[0160] wherein LI, AL, and ALB are as defined for formulas (IIIa) and (IVa), and PL may or may not be present in either or both of the polypeptide of formula (IIIc) or (IIId) and / or the polypeptide of formula (IVc) or (IVd).

[0161] In a specific embodiment, the PL comprises an amino acid sequence between the ISBP and the CE. In a particular embodiment, the AL of the polypeptide of formula (III) and the ALB of the polypeptide of formula (IV) can be bound to each other or are bound to each other.

[0162] In such configurations, 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 probe comprises a first polypeptide comprising an inorganic surface-binding polypeptide and an active linker (AL) and a second polypeptide comprising an analyte-specific capture element, where the AL can bind to the analyte-specific capture element (or a polypeptide comprising a CE). In certain embodiments, the AL binds directly to the analyte-specific capture element, e.g., the AL can be protein A, protein G, or anti-IgG (e.g., goat anti-human IgG), and the analyte-specific capture element can be an antibody or an 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, e.g., the ALB can be biotin and the AL can be streptavidin, or vice versa. In certain embodiments, the protein G is fused to the N-terminus of the inorganic surface-binding polypeptide, e.g., via an inert linker, e.g., a direct bond or a peptide linker. In certain embodiments, streptavidin is fused to the N-terminus of the inorganic surface-binding polypeptide, for example, via an inert linker, for example, a direct bond or a peptide linker. In certain embodiments, protein G is fused to the C-terminus of the inorganic surface-binding polypeptide, for example, via an inert linker, for example, a direct bond or a peptide linker. In certain embodiments, streptavidin is fused to the C-terminus of the inorganic surface-binding polypeptide, for example, via an inert linker, for example, a direct bond or a peptide linker. In addition to biotin and streptavidin, various other binding pairs are known in the art and can be used alternatively.

[0163] In a specific embodiment, the ISBP of the dual affinity probe is selected from the group consisting of a binding peptide, a protein, an antibody having affinity for an 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.

[0164] 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 has a V H In another specific embodiment, ISBP is a V H Gold-binding motif In another specific embodiment, the ISBP is an antibody specific for binding to gold.

[0165] In further specific embodiments, AL is an amino acid sequence comprising protein G from Streptococcus, or an amino acid sequence comprising streptavidin from Streptomyces.

[0166] In alternative embodiments, the linker sequence may comprise other amino acid sequences, such as an inert linker, a linear tandem repeat polypeptide, a linear non-repetitive polypeptide, or a linker that allows for additional flexibility or rigidity to the ISBP or CE.

[0167] In another embodiment, the linker sequence may contain an additional inert linker that binds directly and covalently to the ISBP but binds with high affinity to IgG or biotin incorporated into the capture element.

[0168] In specific embodiments, the inert linker may comprise, for example, the following amino acid sequence: GGGGSGGGGSGGGGSASGGG [SEQ ID NO: 1]

[0169] The inert linker of SEQ ID NO:1 may further be incorporated or fused to another amino acid sequence on the linker, such as streptavidin or a high affinity protein such as protein G (AL). In a specific embodiment, SEQ ID NO:1 is fused directly to protein G to form the following sequence [SEQ ID NO:2]: MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNG VDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSASGGG

[0170] In this example, the inert linker SEQ ID NO:1 is on the C-terminus and directly links the ISBP and the amino acid sequence of protein G which binds with high affinity to a capture element which may be any IgG antibody or a suitable fragment of an IgG antibody.

[0171] In another specific embodiment, an inert linker, such as SEQ ID NO: 1, can be fused to streptavidin at the linker. In a specific embodiment, the inert linker SEQ ID NO: 1 is on the C-terminus and directly linked to ISBP, and the streptavidin amino acid sequence binds with high affinity to biotinylated capture elements.

[0172] In another embodiment, the linker sequence does not include an inert linker. For example, the linker AL may be specific only to the amino acid sequence of protein G, such as SEQ ID NO: 19, a variant thereof, or the streptavidin amino acid sequence.

[0173] 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, the antibody is conjugated with biotin (ALB), and AL is an amino acid sequence comprising streptavidin from Streptomyces. In another embodiment, the CE is an antibody, and 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 probe or immunoprobe is labeled with a detectable label. In certain embodiments of the dual affinity immunoprobe, the polypeptide that comprises the analyte-specific capture element is labeled with a detectable label.

[0174] Sample Composition In a specific embodiment, the dual affinity probe may be included in the composition, or the analyte composition. In a specific embodiment, the analyte composition may include any dual affinity probe described herein. In another specific embodiment, the analyte composition may include one, two, three, four, or five different dual affinity probes described herein. In a specific embodiment, the analyte composition includes a dual affinity probe and / or at least one additional binding moiety, such as an antibody or antigen. In another embodiment, the composition may be included on a surface of the system or diagnostic kit. In another specific embodiment, the composition is added to the surface, thereby allowing the dual affinity probe and / or the binding moiety to flow down the surface. In a specific embodiment, the analyte composition is added to a pretreated sample pad in a lateral flow assay (LFA).

[0175] In a specific embodiment, the analyte composition may not include a dual affinity probe, but does include a binding moiety.

[0176] In another embodiment, the analyte composition comprises 1, 2, 3, 4, 5, 6, 7, or 8 different binding moieties.

[0177] In a specific embodiment, at least one additional binding moiety comprises a surface particle. In another embodiment, at least one additional binding moiety does not bind to a surface particle. In another embodiment, one binding moiety is a dual affinity probe and the second binding moiety does not bind to a surface particle. In a specific embodiment, the second binding moiety comprises an antibody or binding fragment thereof, optionally a non-fragmented antibody, a modified antibody, a camelid antibody, a nanobody, a single chain variable fragment, or a Fab fragment.

[0178] In another embodiment, the additional binding moiety specifically binds to the analyte. In a specific embodiment, the binding moiety is an antibody. In another embodiment, the antibody comprises a tag, such as a biotin tag or a his tag.

[0179] Analyte detection systems and kits In one embodiment of the invention, the dual affinity probe may be utilized and / or included in an analyte detection or diagnostic system, such as a diagnostic kit, hi a specific embodiment, the system or kit is used to detect a specific analyte in a sample.

[0180] In certain embodiments, the system or kit includes an analyte capture element that directly or indirectly binds to the analyte being detected, and a detection element that directly or indirectly binds to the analyte-bound analyte capture element. In certain embodiments, the analyte capture element and the detection element each include a surface-binding moiety, and in certain embodiments, they include surface-binding moieties that bind to different surfaces. In some embodiments, the analyte capture element includes an antibody that binds to the target analyte, and the surface-binding moiety is directly bound or fused to the antibody, optionally via a linker. In some embodiments, the analyte capture element includes an antibody that binds to the target analyte, and the surface-binding moiety is indirectly bound to the antibody, for example, via another binding member, for example, an agent that binds to the antibody Fc or hinge domain. In some embodiments, the detection element includes an antibody that binds to the target analyte, and the surface-binding moiety is directly bound or fused to the antibody, optionally via a linker. In some embodiments, the detection element includes an antibody that binds to the target analyte, and the surface-binding moiety is indirectly bound to the antibody, for example, via another binding member, for example, an agent that binds to the antibody Fc or hinge domain. The use of agents and systems based on indirect binding of analytes advantageously allows the system to be used with a wide variety of different analyte capture and detection elements without the need to attach surface binding moieties to them.

[0181] The system or kit can be used in a sandwich assay to detect an analyte, the system comprising two antibodies that bind to the analyte at different locations, one antibody being a component of the analyte capture element and the other antibody being associated or bound to the detection element. The system and assay can also be modified for competitive assays.

[0182] In certain embodiments, the analyte capture element and / or detection element comprises another binding moiety that binds directly or indirectly to the antibody that binds to the analyte to promote the formation of a complex comprising the analyte capture element and the analyte, or a complex comprising the analyte capture element, the analyte, and the detection element. A variety of binding moieties can be used, including, but not limited to, secondary antibodies, and a variety of binding pairs.

[0183] In one example, the analyte-binding moiety is tagged with a polypeptide sequence that is bound by a polypeptide (e.g., an antibody). Such tag sequences are known and available in the art and include, for example, his-tag, FLAG-tag, biotin-tag, and myc-tag. Such tag sequences can be incorporated into the antibody during production or synthesis of the antibody, for example, as a fusion to the C-terminus of the antibody heavy chain. When the analyte-binding antibody includes a tag, it can be indirectly bound by a capture or detection element that includes a polypeptide that binds to the tag sequence, for example, an anti-biotin or anti-his antibody. In certain embodiments, the detection element includes an anti-tag antibody that includes a surface-binding moiety, for example, fused to the C-terminus of its Fc domain. As another example, one element can include a member of a binding pair, such as streptavidin, avidin, or neutravidin, that binds to a biotin tag.

[0184] As another example, one component may contain a secondary antibody that binds only one of the analyte-binding antibodies, and the other component may contain a secondary antibody that binds only the other analyte-binding antibody. This can be accomplished in a variety of ways, for example, one analyte-binding antibody may contain a human Fc that is bound by a specific secondary antibody, and the other analyte-binding antibody may contain an Fc of a different species that is bound by a different secondary antibody. In other embodiments, one antibody may be IgG and the other may be IgM or IgA, which are bound by different proteins, IgG being bound by protein G, IgM being bound by protein L, and IgA being bound by jaculin.

[0185] The compositions, systems, kits, and methods disclosed herein can be used in lateral flow assays. For example, in certain assays, a detection element is bound to a lateral flow surface, such as nitrocellulose, at a first location. An analyte capture element (or a portion thereof), e.g., in lyophilized form, is present at a different second location on the lateral flow surface. When a test sample is added to the second location (in the liquid), it mixes with the analyte capture element, which binds to the analyte present in the sample. The liquid containing the analyte capture element flows across the lateral flow surface to the first location, where the analyte capture element bound to the analyte is bound by the detection element. In certain embodiments, the analyte capture element is bound or associated with nanoparticles or beads, e.g., gold beads, which facilitate its detection at the second location. Thus, in certain embodiments, the analyte capture element includes a nanoparticle, e.g., gold, surface-binding moiety, which binds to the lateral flow surface, e.g., a cellulose or nitrocellulose surface-binding moiety.

[0186] The assays and systems disclosed herein may employ a variety of different configurations, including but not limited to those specifically described herein or shown in the accompanying examples and figures. For example, in each case, any of the antibodies may instead be a molecularly imprinted polymer (MIP) or an aptamer or an engineered protein.

[0187] In certain embodiments, the systems, reagents, and methods disclosed herein can be adapted to detect small analytes, e.g., analytes that cannot be bound by two different antibodies simultaneously. Examples of such small analytes include, but are not limited to, peptides, toxins, drugs, metal ions, and hormones. In certain embodiments, the small analyte has an MR<1000. In certain embodiments, this is accomplished by using two antibodies to form an antibody sandwich type complex with the small analyte, where a first antibody binds to the small analyte and a second antibody binds to the first antibody only if the first antibody binds to the small analyte. In one embodiment, binding of the first antibody to the small analyte causes a conformational change in the first antibody, thus unmasking the epitope to which the second antibody binds. Antibodies that bind to small analytes can be generated by immunizing animals with the small analyte conjugated to a hapten. A second antibody that binds to the first antibody only if the first antibody binds to the small analyte can be generated or identified by screening an antibody library.

[0188] In a specific embodiment, the system may include a) an analyte capture element comprising an antibody associated with a particle, such as a nanoparticle or bead, which binds to the analyte; b) a sandwich element comprising an antibody that binds to the analyte; and c) a lateral flow capture element that is a fusion polypeptide comprising a first surface binding moiety fused to a binding member. In another embodiment, the analyte capture element is directly or indirectly physisorbed to the nanoparticle or bead. In one embodiment, the analyte capture element is directly or indirectly attached to the nanoparticle or bead, for example via a surface binding moiety. In another embodiment, the analyte capture element is directly or indirectly physisorbed to the nanoparticle or bead. In another embodiment, the analyte capture element is directly or indirectly attached to the nanoparticle or bead. In a specific embodiment, the analyte capture element comprises a second surface binding moiety that is directly attached to the nanoparticle or bead. In another embodiment, the analyte capture element is bound by a secondary binding moiety that comprises a second surface binding moiety that is directly attached to the nanoparticle or bead. In another specific embodiment, the surface binding moiety is bound to cellulose or nitrocellulose. In another embodiment, the lateral flow capture element comprises a surface binding moiety that comprises a polypeptide having a sequence as set forth in Table 1. In another specific embodiment, the surface-binding moiety is fused to an additional polypeptide sequence, thereby having a sequence as set forth in Table 1a.

[0189] In a specific embodiment, the system may comprise: a) a first antibody bound to a nanoparticle surface via a first surface-binding moiety fused to an Fc region of the first antibody, the first antibody binding to an analyte; b) a second antibody comprising a tag polypeptide sequence, optionally a biotin tag polypeptide sequence or a His tag polypeptide sequence, the second antibody binding to the analyte; and c) a third antibody bound to a second surface, optionally a cellulose or nitrocellulose surface, via a second surface-binding moiety fused to an Fc region of the third antibody, the third antibody binding to a tag polypeptide sequence present on the second antibody, wherein the first and second antibodies bind to different epitopes of the analyte and do not compete with each other for binding to the analyte. In another embodiment, the system may include a) a first antibody that binds to the analyte, b) a second antibody that binds to the analyte, where the first and second antibodies bind to different epitopes of the analyte and do not compete with each other for binding to the analyte, c) a third antibody bound to the nanoparticle surface via a first surface-binding moiety fused to an Fc region of the third antibody, where the third antibody binds to the first antibody, and d) a fourth antibody bound to a first region of the surface, optionally a cellulose or nitrocellulose surface, via a second surface-binding moiety fused to an Fc region of the fourth antibody, where the fourth antibody binds to the second antibody but not the first antibody. In another embodiment, the system may further include e) a fifth antibody bound to a second region of the surface via a third surface-binding polypeptide fused to an Fc region of the fifth antibody, where the fifth antibody binds to the first antibody but not the second antibody.

[0190] In another specific embodiment, the detection system can be a detection kit or a diagnostic kit. In a specific embodiment, the kit can be a kit for detecting an analyte, a kit for point-of-care service, a kit for detecting an analyte sample in a hospital / clinical environment, or a kit for detecting an analyte in a sample that can be used at home. In a specific embodiment, the kit can be an assay such as an immunoassay. In another embodiment, the kit can be a dipstick assay. In another embodiment, the kit can be a lateral flow assay. In a specific embodiment, the kit is a diagnostic kit, meaning that the kit can be used in the above environments to detect an analyte in a sample.

[0191] In a specific embodiment, the kit or diagnostic kit comprises: a) a surface; b) a surface binding moiety (SBM) bound to at least a portion of the surface; c) a reagent for collecting a sample; and d) a specimen composition comprising at least one set of dual affinity probes (DAPs).

[0192] In another example, the kit comprises: a) a surface; b) a surface-binding moiety (SBM) bound to at least a portion of the surface, the SBM comprising a surface-binding peptide (SBP) and / or, optionally, a non-analyte capture element (NACE); c) a reagent for collecting a sample; and d) an analyte composition comprising a set of at least one dual affinity probe (DAP), wherein at least one DAP is any one of the dual affinity probes described herein. In a specific embodiment, the dual affinity particles comprise gold surface particles.

[0193] In specific embodiments, the surface of the kit may be one or more selected from the group consisting of PDMS, ZnO, gold, quartz, silica, silicon, silver, cellulose, nitrocellulose, plastic, polystyrene, and graphene. In specific embodiments of the kits herein, the SBP comprises a nitrocellulose binding protein, a silicon binding protein, a cellulose binding motif, a polystyrene binding motif, and / or a silica binding motif. In another embodiment, the SBP comprises a sequence selected from Table 1. In another specific embodiment, the SBP comprises a silicon binding protein of EMT-020 to EMT-025, the cellulose binding motif is cellulose binding motif 1 or cellulose binding motif 2, and the polystyrene binding motif is polystyrene binding motif 1 or polystyrene binding 2, and / or silica binding motif 1.

[0194] In another embodiment of the kit, the SBM comprises one or more NACEs selected from the group consisting of a linker, streptavidin, protein G, biotin, a biotinylated protein, an antigenic protein or peptide, and an antibody, wherein the antibody is an antibody or a binding fragment thereof, optionally a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment.

[0195] In another specific embodiment, the SBM comprises an SBP fused or conjugated to a NACE, wherein the fusion protein is selected from one or more of the group consisting of Table 1a.

[0196] In another embodiment, the analyte composition is any of the analyte compositions described herein. In another embodiment, the analyte composition is provided in lyophilized beads. In another embodiment, the kits herein include at least 2, 3, 4, or 5 analyte compositions.

[0197] In a specific embodiment, the kit further comprises at least a second analyte composition comprising a second analyte binding moiety (SABM), where the SABM may comprise one or more selected from a surface particle, an SBP, a NACE, and a CE. In an embodiment, the second analyte composition may comprise any of the dual affinity probes described herein. In a specific embodiment, the SABM comprises a NACE, where the NACE is one or more selected from the group consisting of a linker, streptavidin, protein G, biotin, an antigenic protein or peptide, and an antibody, where the antibody is an antibody or binding fragment thereof, optionally a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment.

[0198] In a specific embodiment, the second analyte composition comprises surface particles as lyophilized analyte beads. In another embodiment, the second analyte composition comprises surface particles bound to SBPs fused or bound to non-analyte capture elements (NACEs), or surface particles fused or bound to NACEs. In a specific embodiment, the NACEs are one or more selected from the group consisting of linkers (LIs), streptavidin, protein G, biotin, antigenic proteins or peptides, and antibodies, wherein the antibodies are antibodies or binding fragments thereof, optionally non-fragmented antibodies, modified antibodies, camelid antibodies, single chain variable fragments, or Fab fragments.

[0199] In a particular kit, the analyte composition comprises a dual affinity probe as described herein, and the second analyte composition comprises a SABM. In a specific embodiment of the kit, both compositions are incorporated into the surface of the kit. In another embodiment, the compositions are located in the same area of ​​the kit, or adjacent or proximate to each other on the kit. In a specific embodiment, there is only a spacer, i.e., a surface separating the two compositions. In a specific embodiment, the composition is located on the surface of a pre-treated sample pad, and comprises a surface selected from one or more selected from the group consisting of PDMS, ZnO, gold, quartz, silica, silicon, silver, cellulose, nitrocellulose, plastic, polystyrene, and graphene.

[0200] In another embodiment, the surface of the kit may include a control surface binding moiety. In a specific embodiment, the control surface binding moiety allows a positive or negative control indication for a user of the kit. In a specific embodiment, the analyte composition or the second analyte composition includes a control binding moiety that binds to the control surface binding moiety. The control surface binding moiety can be tested adjacent or in close proximity to a line or region containing SBP, thereby providing a control near the binding of DAP to the surface-bound surface binding moiety. In a specific embodiment, the control binding moiety in the analyte composition or the second analyte composition includes a surface particle. In another embodiment, the control binding moiety in the analyte composition or the second analyte composition includes SBP or NACE bound to a surface particle. In another embodiment, the control binding moiety in the analyte composition or the second analyte composition includes a tag, e.g., biotin, a his tag, or protein G. In another embodiment, the control binding moiety includes an antibody, the antibody being an antibody or a binding fragment thereof, optionally a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment. In another embodiment, the control binding moiety in the analyte composition or the second analyte composition comprises a control antibody or a surface particle bound or fused to a control binding antibody, such as an IgY antibody, where the antibody is an antibody or binding fragment thereof, optionally a non-fragmented antibody, a modified antibody, a camelid antibody, a single chain variable fragment, or a Fab fragment. In another embodiment, the control surface binding moiety binds to the control binding moiety to indicate that the assay has been performed as properly performed, for example, in a positive control. For example, in one embodiment, the DAP, when used in an assay, may comprise a biotin-tagged moiety that binds to streptavidin bound, fused, or immobilized to the surface of the assay. Thus, in one embodiment, the control surface binding moiety and the control binding moiety are designed to bind to each other, such as in the following binding relationships: 1) biotin-streptavidin, 2) protein G-IgG antibody, and 3) anti-IgG antibody-IgG antibody.

[0201] In another embodiment of the kit, the kit may be in the form of a lateral flow assay. In a specific embodiment, the LFA comprises a) a surface, b) a surface-binding moiety (SBM) bound to at least a portion of the surface, c) a reagent for collecting a sample, and d) an analyte composition comprising at least one set of dual affinity probes (DAPs). In another embodiment, the LFA may be used in the form as exemplified in Examples 20-27 herein.

[0202] An exemplary assay utilizing a (nitro)cellulose-bound streptavidin fusion protein (and, for example, a dual affinity probe as described herein) for detecting or quantifying analyte levels in a test sample, such as SARS-CoV-2 nucleocapsid or spike protein. The assay is manufactured into a diagnostic kit using the dual affinity probe according to the methods described above. The kit may have a pre-treated sample pad portion for collecting and reacting a biological sample from a subject, such as a human, a surface (e.g., a nitrocellulose membrane portion where the detection complex is formed on the test line), and an absorbent pad at the end of the strip for collecting the flow-through. In a specific embodiment, the pre-treated sample pad contains an analyte composition including DAP for binding to the analyte. The sample pad is treated with the analyte composition immediately prior to use of the kit, or alternatively, beads are added during manufacture of the kit.

[0203] A test sample having an analyte of interest can be collected from a subject using various collection methods (e.g., collecting a spat sample in a tube, collecting a nasal swab using an assay strip, etc.), and the collected test sample is introduced into a reagent solution. The reagent solution containing the test sample is then applied directly onto the pre-treated sample pad. The sample containing the analyte flows laterally over the surface and then contacts an analyte composition containing DAP, e.g., DAP beads are dissolved in the sample reagent and bind to the analyte. The analyte-bound DAP then flows laterally over the surface of a surface such as a nitrocellulose membrane. In this case, the analyte-bound DAP is captured at the test line by forming a complex (i.e., a detection conjugate) with an immobilized surface-binding moiety bound to the surface. The presence of the detection conjugate captured at the test line is observed with the naked eye to determine the presence and / or amount of the analyte in the test sample.

[0204] In a specific embodiment, the LFA can utilize a kit or system described herein. In another embodiment, the LFA can utilize a DAP, an additional binding moiety, or an analyte composition, or a further open analyte composition described herein. In a specific embodiment, the increase in signal provides a method for detection or quantification of the analyte in the sample.

[0205] In specific embodiments, the LFA kit may include one or two analyte compositions. In another embodiment, the composition may use gold surface particles such as Examples 20-27 described herein. In specific embodiments, the LFA kit may utilize antigens or recombinant antigens in one or more analyte compositions. In specific embodiments, the use of antigens is for the DAP to compete with the analyte in the sample. In specific embodiments, the analyte in the sample outcompetes the antigen in the analyte composition for binding to the DAP. In such a system, binding of the analyte reduces binding of the antigen, thereby allowing for a competitive assay in which the signal increases or decreases based on the amount of analyte in the sample. In specific embodiments, the analyte binds to the binding moiety without the surface particle, thereby reducing the signal of the assay. In other words, the reduction in signal provides a method for detection or quantification of the analyte in the sample.

[0206] Methods for detecting analytes The present disclosure also provides a method for determining the presence and / or quantification of an analyte in a test sample, comprising: contacting a test sample with a dual affinity probe comprising an SBM, e.g., an inorganic surface binding peptide (ISBP) and an analyte-specific capture element, under conditions and for a time sufficient to allow an analyte present in the test sample to bind to the analyte-specific capture element, thereby forming a complex comprising the analyte bound to the dual affinity probe; determining the presence and / or amount of the complex and / or analyte present in the complex; the presence of the complex and / or analyte indicates the presence of the analyte in the test sample, and the amount of the complex and / or analyte indicates the amount of analyte present in the test sample; Thereby, a method is provided for determining the presence of and / or quantifying an analyte in a test sample.

[0207] In some embodiments, the test sample is a biological sample, such as a biological sample obtained from a subject, such as, for example, 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, such as a human. In some embodiments, the biological sample comprises pathogens, antibodies, cells, and / or other biomolecules. The method may be used to test a variety of different types of samples, including, for example, environmental samples (including samples collected in the built environment), water, or food or beverage samples, etc.

[0208] The disclosed method can 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 to 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 pathogen is a virus, a bacterium, a fungus, a protozoan, an insect, or a prion. In certain embodiments, the virus is an influenza virus or a coronavirus, such as the SARS-CoV-2 virus. In other embodiments, the analyte is an antibody that specifically binds to one or more infectious agents or pathogens.

[0209] The method may also use a capture element that is not an antibody or antibody fragment, but is any amino acid sequence, peptide, protein, or specific antigen that binds to an antibody from a pathogen. For example, a capture element can be used to test a biological sample obtained from a subject to determine whether the subject has antibodies to a specific pathogen, or more specifically, a specific antigen or epitope that identifies the pathogen. In a specific embodiment, the capture element comprises an antigen or an epitope thereof. For example, a biotinylated SARS-CoV-2 spike protein antigen may be conjugated to a streptavidin fusion protein for detection of spike protein-specific antibodies in a test sample.

[0210] The capture element may be specific for any analyte or pathogen of interest, for example, the capture element may be specific for an antigen, protein, peptide, antibody or antibodies, or other organic moiety that identifies a subject as positive for or susceptible to a particular pathogen. In certain embodiments, the capture element is specific for an antibody that specifically binds to the 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 a SARS-CoV-2 spike (S) antigen or a SARS-CoV-2 nucleocapsid (N) antigen or variants thereof.

[0211] In various embodiments, the analyte-specific capture element specifically binds to an analyte of interest to determine whether the analyte of interest is present in the test sample and / or the amount or concentration thereof present in the test sample. In certain embodiments, the analyte-specific capture element comprises an antibody or antigen-binding fragment thereof specific for a pathogen or an antigen thereof, such as a SARS-CoV-2 spike (S) antigen or a SARS-CoV-2 nucleocapsid (N) antigen.

[0212] In certain embodiments, the inorganic surface-binding peptide comprises one or more gold-binding peptides, silver-binding peptides, silica-binding peptides, plastic-binding peptides, cellulose-binding peptides, or graphene-binding peptides, including but not limited to any of the peptides in Table 1 herein.

[0213] In certain embodiments, the dual affinity immunoprobe binds to the inorganic surface via the inorganic surface-binding peptide and the test sample when the test sample contacts the dual affinity immunoprobe. One example is a lateral flow assay. However, in other embodiments, the dual affinity immunoprobe does not bind to the inorganic surface when the test sample contacts the dual affinity immunoprobe. For example, the dual affinity immunoprobe and the test sample may be contacted in a solution to form a complex, and then the solution is contacted with an inorganic surface, thereby binding the dual affinity immunoprobe to the inorganic surface. In certain embodiments, the inorganic surface is a biosensor material selected from the group consisting of gold, silica, silver, cellulose, plastic, and graphene. The bound complex or analyte can be detected and / or quantified by various means, for example, using a quartz crystal microbalance, surface plasmon resonance (SPR), or lateral flow.

[0214] In various embodiments, the methods may employ the use of one or more positive or negative controls, 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 to the analyte of interest.

[0215] In certain embodiments, an analyte is determined to be present in a test sample when it is detected in the test sample or when it is determined to be present in a certain level or amount in the test sample.For example, the level or amount indicating the presence of an analyte in a test sample may be a predetermined amount based on previous experience, or may be an amount greater than the amount determined using a negative control, for example, at least 10%, at least 20%, at least 50%, at least 2-fold, or at least 3-fold greater than the amount determined in a negative control.

[0216] In a specific embodiment, the detection of the analyte, i.e., confirmation that the subject is positive for the analyte, can be determined by a binding curve, such as by SPR or QCM-D. In other words, the presence of the analyte is determined, for example, by obtaining a specific RU or other response or detection curve. In another embodiment, the presence of the analyte is determined by color contrast with a negative control. Such contrast can be determined by visually assessing the individual as instructed in the assay instructions. Such determination can be made at the point of care, in a hospital, or at other medical facilities. In another embodiment, the presence of the analyte is determined by color contrast with a negative control by a device such as a multi-well plate color reader.

[0217] The accompanying examples illustrate specific embodiments of the compositions and methods disclosed herein.

[0218] Directional loading of an antibody onto an inorganic binding entity was achieved in one embodiment by adsorbing it to proteins A and G, which contain binding domains for the Fc (fragment crystallizable) region of the antibody.

[0219] In other embodiments, directional immobilization of recognition biomolecules (eg, capture elements) is achieved using the streptavidin-biotin system, which represents one of the strongest non-covalent interactions in nature.

[0220] In another embodiment, the fusion protein containing the inorganic binding peptide is linked to a single chain variable fragment (scFv) or Fab fragment or full-length antibody against the pathogen of interest. These methods can be used in engineering the dual affinity immunoprobe of the present invention. Reversible and irreversible binding methods of antibodies known in the art are described in detail in (MAKARAVICIUTE;RAMANVIEN, 2013) and (LIEBANA;DRAGO, 2016).

[0221] Inorganic surface-binding peptides can include those that specifically bind to gold, silica, and graphene, as well as cellulose, silver, and carbon-based synthetic polymers (plastics).

[0222] Sensor types can include, for example, 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. Biosensor materials include, for example, gold, silver, silica, graphene, cellulose, and carbon-based synthetic polymers.

[0223] Pathogens may include, by way of example, coronavirus species, such as SARS and MERS, influenza species, respiratory syncytial virus species, adenovirus species, parainfluenza species, filoviridae, such as Ebola and Marburg, hantavirus species, arenaviridae, such as Lassa, bunyaviridae, such as Rift Valley and Crimean-Congo, and paramyxoviridae, such as Hendra and Nipah. Pathogens, in some embodiments, include prions. Pathogens, in some embodiments, include gram-negative and gram-positive bacteria.

[0224] Antibody types can include, but are not limited to, humanized, monoclonal, polyclonal, and synthetic antibodies.

[0225] Detection methods using the dual affinity immunoprobes of the present invention include, but are not limited to, lateral flow in a multi-well plate color reader, dipstick color change, SPR, and quartz crystal microbalance with dissipation monitoring (QCM-D).

[0226] In a specific embodiment, the detection method may use any of the systems or kits described herein. In a specific embodiment, the method includes a method of determining the presence of an analyte and / or quantifying an analyte in a sample from a subject by testing the sample with any one of the diagnostic kits or systems described herein, the method includes a) contacting the test sample with a reagent, b) applying the test sample with the reagent to a surface, thereby flowing the test sample laterally over the surface, thereby contacting the DAP composition on the surface, c) directly binding the analyte present in the test sample to the CE, thereby forming a complex containing the analyte bound to the DAP, d) further flowing the analyte complexed with the DAP laterally over the surface, thereby complexing it with a surface binding moiety (SBM), and e) determining the presence and / or amount of the analyte in the test sample by the presence of the DAP complexed to the SBM. In a specific embodiment, the surface containing the DAP composition is a pre-treated sample pad. In another embodiment, the test sample in the reagent is added to a pre-treated sample pad. In another specific embodiment, the pre-treated sample pad has a different surface membrane than the surface containing the surface binding moiety (SBM). In another specific example, the pre-treated sample pad includes a second analyte composition that includes SABMs, allowing the test sample to flow laterally over the surface and bind to the SABMs in the second analyte composition.

[0227] In another embodiment, the method may include determining the presence of an analyte and / or quantifying an analyte in a sample from a subject by testing the sample with any one of the diagnostic kits described herein, the method comprising: a) contacting the test sample with a reagent; and b) applying the test sample along with the reagent to a surface, thereby causing the test sample to flow laterally across the surface, thereby contacting the test sample with a DAP composition on the surface, the DAP composition comprising i) surface particles fused or bound to an antigen, and ii) an anti-analyte antibody that binds to the antigen; and b) determining the presence of the analyte in the test sample and the surface particles fused or bound to the antigen. and c) competitively binding to the anti-analyte antibody, thereby forming two complexes, where the anti-analyte antibody is complexed to the surface particle fused or conjugated to the analyte and / or antigen in the test sample, forming a complex containing the analyte bound to the DAP, d) the presence of more analyte in the sample means that the anti-analyte antibody is mostly complexed to the analyte in the test sample and little to the gold particle fused or conjugated to the antigen, and e) the sample flows further laterally across the surface, where the SBM binds to the tag on the anti-analyte antibody, thereby causing the anti-analyte antibody to specifically conjugate to the surface binding moiety (SBM) on the surface. In a specific embodiment, the tag is a his-tag or a biotin tag. In a specific embodiment, the surface particle is a gold particle, fused or conjugated to the antigen, and the gold particle and the anti-analyte antibody are in separate compositions, and the two compositions are applied adjacently on the surface. In another embodiment, the second composition comprising the anti-analyte antibody further comprises a control antibody fused or conjugated to a gold particle, and after the sample is further subjected to lateral flow over the surface, the control antibody fused or conjugated to the gold particle binds to the anti-control antibody on the surface, thereby providing a positive control for the diagnostic kit.

[0228] The methods may also include a method of determining the presence of an analyte in a sample from a subject and / or a method of quantifying an analyte by testing the sample with any one of the diagnostic kits described herein, comprising: a) contacting the test sample with a reagent; b) applying the test sample along with the reagent to a surface, thereby causing the test sample to flow laterally across the surface, thereby contacting the DAP composition on the surface (the DAP composition comprises i) gold particles fused or conjugated to an antigen, and ii) an anti-analyte antibody that binds to the antigen); c) allowing the analyte present in the test sample and the gold particles fused or conjugated to the antigen to compete with the anti-analyte antibody. Specifically, the method comprises: specifically binding the anti-analyte antibody to the gold particles fused or conjugated to the analyte and / or antigen in the test sample; d) thereby forming two complexes (the anti-analyte antibody is conjugated to the gold particles fused or conjugated to the analyte and / or antigen in the test sample to form a complex containing the analyte bound to the DAP); e) further flowing the complex laterally over the surface and contacting it with a second composition comprising an anti-IgG antibody, thereby binding the anti-IgG antibody to the anti-analyte antibody; and f) further flowing the sample laterally over the surface, the SBM binds to the anti-analyte antibody or a tag on the anti-IgG antibody, thereby specifically conjugating the anti-analyte antibody to the surface binding moiety (SBM). In a specific embodiment, the tag is a his-tag or a biotin tag. In another embodiment, the tag is on the anti-IgG antibody. In another embodiment, the second composition comprising the anti-IgG antibody further comprises an IgY antibody fused or conjugated to the gold particles, and after the sample is further flowed laterally over the surface, the IgY antibody fused or conjugated to the gold particles binds to the anti-IgY antibody, thereby providing a positive control for the diagnostic kit.

[0229] In another embodiment, the method may include the test sample being 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, or a combination thereof. In another embodiment, the analyte is a pathogen. In a specific embodiment, the pathogen is a virus, a bacterium, a fungus, a protozoan, an insect, or a prion. In a specific embodiment, the virus is a SARS-CoV-2 virus. In another specific embodiment, the analyte is a SARS-CoV-2 spike (S) antigen or a SARS-CoV-2 nucleocapsid (N) antigen, or an antibody specific for binding to a SARS-CoV-2 spike (S) antigen or a SARS-CoV-2.

[0230] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the present invention and not to limit its scope.

[0231] Acronyms or short forms used in the examples

[0232] H=time

[0233] Min=minutes

[0234] s=second

[0235] PBS = phosphate buffered saline

[0236] E. coli = Escherichia coli

[0237] SARS-CoV-2 = Severe Acute Respiratory Syndrome Coronavirus 2

[0238] BSA = bovine serum albumin

[0239] ddH2O = double distilled water

[0240] Example 1 Common methods Synthetic peptide identification and synthesis: Six gold-binding peptides and six silica-binding peptides from the literature were custom synthesized to >90% purity using FMOC (fluorenylmethyloxycarbonyl chloride) synthesis (Pierce ThermoFisher).

[0241] Fusion protein design: The general structure of an embodiment of the present invention is inorganic surface binding peptide + linker + protein G', a known version of protein G with the albumin binding site removed (a version of the Uniprot Q54181 protein). The amino acid sequence of protein G' plus the linker [SEQ ID NO:2] is as follows:

[0242] MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNG VDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSASGGG

[0243] Antibodies and antigens: Monoclonal antibodies against 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).

[0244] Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) for Comparative Peptide Bond Analysis:

[0245] The Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) is an instrument that measures mass and viscosity at or near surfaces and in thin films. QCM-D is able to detect extremely small chemical, mechanical and electrical changes that occur on the sensor surface and convert them into interpretable electrical signals (TONDA-TURO; ARMAGNOLA; CIARDELLI, 2018).

[0246] All QCM-D analyses were performed on a 4-channel Qsense™ analyzer (Biolin Scientific, Gothenburg, Sweden) at 23°C. Gold and silica Qsense™ sensor chips were rinsed with 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 PBS. Gold or silica sensor chips were loaded onto the instrument and allowed to equilibrate for 15 min. 10 mM PBS was then added to measure frequency and dissipation D Δf n The flow rate was 50 μL / min until equilibrium.

[0247] The gold- and silica-bound peptides were flowed over the gold and silica sensors for 1 h, followed by a 30 min washing step with 10 mM PBS. Raw data were analyzed using the Kelvin-Voigt viscoelastic model with Qsense™ Dfind™ analysis software.

[0248] Surface plasmon resonance (SPR) analysis

[0249] Surface plasmon resonance occurs when polarized light strikes a metal film at the interface of media with different refractive indices. SPR technology works by exciting and detecting the collective oscillation of free electrons, whereby light is focused through a glass prism onto the metal film and its reflection is detected. At a certain angle of incidence (or resonance angle), the electrons (aka plasmons) are set to resonate and absorb the light at that angle. This causes a dark line in the reflected beam.

[0250] The resonance angle can be determined by observing the SPR reflection intensity. The shift in the reflectance curve represents a molecular binding event occurring on or near the metal film, or a conformational change in a molecule bound to the film. The shift versus time provides information about the molecular binding event and the binding kinetics.

[0251] All SPR experiments were performed at 25°C on an 8-channel Biacore™ 8K instrument (Cytiva Lifesciences (formerly GE Healthcare Lifesciences)), Marlborough, MA, USA) using 2xHBS-EP+ running buffer and chips from the Biocore™ SIA AU kit (Cytiva Lifesciences).

[0252] Example 2 Production and purification of gold- and silica-binding fusion proteins in Escherichia coli: The protein sequences of the fusion proteins, containing a well-described gold-binding (BROWN, 1997) and silica-binding (ETESHOLA; BRILLSON; LEE, 2005) peptide fused to a linker and protein G' from Streptococcus, were converted to cDNA with codon usage specific to E. coli. An N-terminal 6x histidine tag was added to the protein for purification purposes. The cDNA inserts representing the fusion proteins were cloned in frame into the E. coli pET-30a(+) expression vector. Correct clones were identified for protein expression using standard molecular cloning techniques. (SAMBROOK; FRITSCH; MANIATIS, 1989) Recombinant proteins were isolated from the supernatant of 1 L of expression culture following a four-step purification protocol including Ni column, TEV protease digestion, Ni column, and finally Q Sepharose column (all reagents were obtained from Genscript, Piscataway, NJ). Protein purity was estimated by densitometric analysis of Coomassie blue-stained SDS-PAGE gels, and endotoxin levels were assessed using an LAL endotoxin assay kit (Xiamen Bioendo Technology Co., Ltd., Xiamen, Fudin, China).

[0253] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0254] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9]

[0255] As shown in Figure 1, 90% purity was achieved for the fusion protein and the ISBP-free G' protein. In Figure 1, three gels A), B) and C) show the expression and purity of the gold- and silica-bound fusion proteins on Coomassie-stained SDS-PAGE gels. 2 μg of BSA was added to lane 1 of each gel A), B) and C) as a loading control. Gel A) shows the ISBP-free fusion protein, gel B) shows the complete gold-bound fusion protein, and gel C) shows the complete silica-bound fusion protein.

[0256] Example 3 Functionalization of QCM-D gold sensors with gold-binding fusion proteins and testing with 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 proteins of Example 2 at 50 μg / mL in PBS were added to obtain a Δf n was flowed over the sensor chip at 50 μL / min until equilibrated, after which the sensor chip was washed with PBS followed by a blocking step with BSA (50 μg / mL in PBS).

[0257] SARS-CoV-2 spike protein antibody was then flowed over the sensor chip at 50 μL / min, followed by a PBS washing step, and finally SARS-CoV-2 spike antigen (50 μg / mL) or negative control (SARS-CoV-2 nucleocapsid antigen, 50 μg / mL) was flowed until the sample was consumed. The sensor was washed with PBS buffer to eliminate non-specific binding. Raw data was analyzed using the Kelvin-Voigt viscoelastic model with Qsense™ Dfind™ analysis software.

[0258] The gold-binding fusion protein was found to bind to the gold sensor surface in two experiments, forming layers of 10.56 nm and 10.5 nm, respectively, with only a small fraction washed off during the subsequent washing steps (residual layer thicknesses were 9.66 nm and 9.6 nm, respectively). Subsequent blocking and washing steps with BSA did not result in any significant changes in layer thickness or mass. SARS-CoV-2 spike protein antibodies were then flowed across the biolayer, more than doubling the thickness and mass of both layers. After a second wash with PBS, biolayers of 20.45 nm (Figure 2, left) and 20.3 nm (Figure 2, right), respectively, remained. To test the ability of the immobilized antibodies to bind antigens and their specificity, SARS-CoV-2 spike antigen (Figure 2, left) and SARS-CoV-2 nucleocapsid antigen (Figure 2, right) were tested in each system. SARS-CoV-2 spike antibodies immobilized on a gold sensor via a gold fusion protein appeared to bind to the spike antigen, forming a 25.29 nm layer after washing with PBS, but did not bind to the nucleocapsid antigen, leaving only a 20.4 nm layer after washing with PBS (compared to the antibody-only layer).

[0259] Example 4 Assessment of the binding kinetics of SARS-CoV-2 spike antibodies binding to gold-conjugated fusion proteins and their ability to bind to the spike antigen: Surface plasmon resonance (SPR), an optoelectronic biosensing technique, was chosen to assess the binding kinetics of spike antibodies to gold fusion proteins bound to gold sensors. First, the immobilization of gold-conjugated fusion proteins and two controls (ISBP-free fusion protein and buffer only) was evaluated. Zero or minimal binding was observed for those controls (Figure 3). However, the gold-conjugated fusion protein showed a five-fold increase in resonance units (RU) during the immobilization step compared to the ISBP-free version. Even after injection of the regeneration buffer, a significant amount of gold-conjugated protein remained immobilized on the gold sensor, indicating that the coated sensor may be reusable.

[0260] The ability and binding kinetics of SARS-CoV-2 spike and nucleocapsid antibodies to bind to the gold-conjugated fusion protein immobilized on the sensor surface, as well as the respective antigens binding to the antibodies, were tested using a dilution series. For both antibodies (Figure 4) and spike antigen (Figure 5), a dilution series was performed across the following concentrations in two experiments: 1.5625 nM (x2), 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. The best concentration for antibody loading was empirically determined to be 2 μg / mL, which was used as the basis for the antigen dilution series. The raw data was analyzed using Biacore™ 8K Evaluation Software version 1.1. As shown in Figure 4, the SARS-CoV-2 spike antibodies bind to the fusion protein. The binding kinetics results for both SARS-CoV-2 spike and nucleocapsid antibodies binding to the fusion protein are shown in Table 2.

[0261] [Table 3]

[0262] Table 2 shows the binding kinetics of spike protein antibody and nucleocapsid antibody to the gold-conjugated fusion protein. The kinetics of the interaction were calculated to give dissociation constants (K) of 1.92E-10 M and 2.58E-10 M for SARS-CoV-2 spike and nucleocapsid antibodies, respectively. D ) was found, which is in line with the K reported in the literature indicating that protein G binds to all human IgG subclasses of approximately 2E-10M. D Similar to QCM-D, these results show that the gold-binding fusion protein efficiently binds to the gold sensor surface and immobilizes and orients the SARS-CoV-2 spike and nucleocapsid antibodies. Second, the SARS-CoV-2 S protein antigen also exhibits a K of 2.39E-9M, which is in the typical range for monoclonal antibody / antigen interactions. D5, indicating that the bound spike protein antibody was able to maintain its antigen-binding affinity (FIG. 5). The results are also shown in Table 3 herein.

[0263] [Table 4]

[0264] Example 5 Conjugation of gold-conjugated fusion proteins to gold nanoparticles: Conjugation of gold-conjugated fusion proteins and ISBP-free fusion protein controls to 40 nm gold nanoparticles (Cytodiagnostics) was tested in 10 mM PBS buffer using increasing amounts of protein (0, 1, 2, 4 µg per 100 µL of 1 OD gold) and increasing pH conditions (5.7-9.8). The results are shown in Figure 6 (hand-drawn section dividers separate the ISBP-free fusion proteins (top four rows) from the gold-conjugated fusion proteins (bottom rows)).

[0265] Scale-up conjugation reaction of gold-conjugated fusion protein: The pH of 1 mL of 40 nm standard gold nanoparticles was adjusted by adding 40 μL of 0.1 M sodium phosphate (pH 6.5). A 10 μg aliquot of the fusion protein was transferred to another microcentrifuge vial and diluted with ddH2O to a total volume of 100 μL. The pH-adjusted gold nanoparticles were quickly added to the vial of diluted fusion protein and incubated at room temperature for 30 min. 50 μL of 10% (w / v) BSA was added to the gold fusion protein mixture and incubated for 5 min to block. The conjugation mixture was centrifuged at 1600×g for 25 min and the supernatant was removed. Finally, the gold conjugate pellet was resuspended in 1xPBS, 1% BSA to a final concentration of OD=5.5 and stored at 4°C until use.

[0266] Example 6 Comparative binding analysis of synthetic peptides to gold and silica sensors using QCM-D: Six gold-binding peptides and six silica-binding peptides described in the literature as binding to gold and silica and shown in Table 1 were synthesized. Their ability to bind to gold and silica sensors was tested using a quartz crystal microbalance with dissipation monitoring (QCM-D). The thickness, deposited mass, elasticity, and viscosity of the layers obtained after PBS washing were calculated. These are summarized in Table 4.

[0267] [Table 5]

[0268] Table 4 summarizes comparative binding experiments of six gold-bound dual affinity probes (EMT014–EMT019) and six silica-bound dual affinity probes (EMT020–EMT025) using a quartz crystal microbalance with dissipation monitoring (QCM-D). Total peptide mass (ng / cm 2 ), molar mass (μmol / m 2 ), thickness (nm), elasticity (kPa) and viscosity (mPa s) are reported.

[0269] The longest gold-binding peptide, EMT015, was found to have the largest mass (ng / cm) deposited on the gold sensor. 2 ) was the largest, but the shortest gold-binding peptide, EMT019, showed a small loading (molar mass (μmol / m 2 It was shown that the fold change (denoted as ) was the largest. The adjusted measurements are a better indicator of the degree of binding. EMT015 formed the thickest layer at 5.152 nm, followed by EMT019, the second highest at 4.48 nm. The layer formed with EMT015 also showed high elasticity and viscosity compared to the other peptides. Of the silica-bound peptides, EMT022 showed the highest mass and molar mass deposited on the silica sensor at a thickness of 4.1 nm compared to the other peptides. It also showed the highest viscosity and second highest elasticity.

[0270] Example 7 Dot Blot Dipstick Assay: Immobilization of antibodies to gold nanoparticles coated with gold-conjugated fusion proteins, as well as their antigen-binding ability, was tested using a dot blot dipstick assay for SARS-CoV-2 spike and nucleocapsid antigens. Amounts of 0.5 μg each of S protein and N protein antigens (diluted in 10 mM sodium phosphate buffer, pH 7.4) were applied to nitrocellulose dipsticks. The dipsticks were then incubated with 80 μL of sample buffer (1xPBS (pH 8), 5% BSA, 0.5% casein, 0.2% Tween 20, 1% PEG 8000), 10 μL of OD5.5 conjugate (prepared as above), and 0.135 μg (in 1 μL) of each antibody at room temperature for 20 minutes. The results are shown in the photographs in Figure 7.

[0271] SARS-CoV-2 spike or nucleocapsid antibodies conjugated to gold nanoparticles via gold fusion protein were able to bind to the spike or nucleocapsid antigens attached to the dipstick when wicked along the nitrocellulose membrane (strips 3 and 4). More antibodies appeared 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 membrane (strips 1 and 2).

[0272] Example 8 Liquid chromatography-tandem mass spectrometry (LC-MS / MS) sequence coverage analysis:

[0273] Proteins are first digested into peptides by a suitable enzyme such as trypsin. Then, the peptide mixture is separated by liquid chromatography. Finally, the MS1 and MS2 spectra of each peptide are detected by mass spectrometry.

[0274] The biological analysis software matches the observed MS1 and MS2 spectra with the theoretical ones to identify each peptide of the protein and then calculates the peptide (or amino acid) coverage percentage.

[0275] Sample preparation

[0276] 50 μL of protein sample was diluted with 50 mM Tris-HCl to prepare a final concentration of 0.2 mg / mL. 0.1 M DTT was then added at a volume ratio of DTT to protein of 1:20 to reduce disulfide bonds. Trypsin was then added at a mass ratio of trypsin to protein of 1:40 to digest for 6 h.

[0277] Finally, the peptides were dried and rediluted using 20 μL of 0.1% FA-H2O for UPLC-MS analysis. UPLC Separation:

[0278] Column temperature: 50°C, flow rate: 300 μL / min, mobile phase: Solvent A: 0.1% FA-2% ACN aqueous solution, Solvent B: 0.1% FA-90% ACN aqueous solution

[0279] Electrospray voltage, 3.5 kV; m / z scan range, 200-2000; ion transfer tube temperature, 333 °C; AGC, 2e5; MS resolution, 120000; collision energy, 32 eV; MS / MS resolution, 15000; threshold ion count, 20000 ions / sec.

[0280] BioPharma™ Finder™ 3.0 was used for LC-MS / MS data analysis. Results: Sequence coverage was 94.0% for the ISBP-free fusion protein (Figure 1A), 95.85% for the gold-bound fusion protein (Figure 1B), and 94.3% for the silica-bound fusion protein (Figure 1C). Sequence coverage simply indicates the percentage of protein that was sequenced using the LC-MS / MS method. LC-MS / MS analysis confirmed the amino acid sequences of the proteins and indicates that the ISBP-, gold-, and silica-bound fusion proteins were expressed as expected.

[0281] Example 9 Comparative binding analysis evaluating direct binding of gold fusion proteins to gold sensors versus EDC-NHS conjugation to conventional gold sensors.

[0282] Immobilization of gold-binding fusion proteins, 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 reference samples on a gold sensor chip using direct immobilization (Figure 8) and EDC-NHS conjugation (Figure 9) techniques, was evaluated by SPR (portable 4-channel P4SPR device, Affinite Instruments).

[0283] As shown below in Table 5, the gold-bound fusion protein exhibited a three-fold increase in resonance units (RU) during the immobilization step with direct binding (2300 RU) compared to the EDC-NHS process (750 RU). These results indicate that direct immobilization to gold is significantly more efficient than immobilization using the EDC-NHS process.

[0284] [Table 6]

[0285] Example 10 Evaluation of the sensitivity and limit of detection (LoD) for 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 proteins on gold sensors prepared by direct immobilization or EDC-NHS conjugation:

[0286] First, gold sensors immobilized with gold fusion proteins by direct binding or EDC-NHS techniques according to Example 9 were conjugated with SARS-CoV-2 spike or nucleocapsid antibodies, followed by SARS-CoV-2 spike antigen or negative control (SARS-CoV-2 nucleocapsid antigen) according to the methods outlined in Example 3.

[0287] Surface plasmon resonance (SPR) was used to evaluate the sensitivity and LoD for binding of SARS-CoV-2 spike protein antigen and SARS-CoV-2 nucleocapsid protein antigen to SARS-CoV-2 spike or nucleocapsid antibody conjugated to gold fusion protein immobilized on gold sensor by direct binding or EDC-NHS immobilization technique of Example 9. As shown in Figure 10, EDC-NHS immobilization technique (left panel) affects the sensitivity and LoD. Approximately 2-fold increase in sensitivity was observed for spike antigen (indicated by S in Figure 10) and approximately 1.3-fold increase in sensitivity was observed for nucleocapsid antigen (indicated by NC in Figure 10). These results show that gold-conjugated fusion protein EMT003 immobilized by direct binding to gold sensor was better than conventional SPR, especially in detecting spike protein.

[0288] Next, detection of nucleocapsid antigen was evaluated using the direct binding EMT-003 gold fusion protein-based SPR system in saliva (human, pooled). As shown in Figures 11A and 11B, binding of recombinant nucleocapsid antigen was visible at all dilutions. Detection was highest in saliva at 1:2 in running buffer.

[0289] Example 11 Detection of SARS-CoV-2 spike protein by SPR: In this example, the performance of EMT003 conjugated to an antibody for selective detection of an antigen under SPR was evaluated. Specifically, EMT003 conjugated to a SARS-CoV-2 anti-spike protein antibody was evaluated for selective detection of the spike protein. EMT003 was diluted to 10 μg / mL. A gold-coated sensor for SPR was then loaded into the flow module in the instrument. 500 μL of distilled water and 500 μL of PBS were flowed briefly over the sensor to establish a baseline signal. The fusion protein EMT003 was then flowed over the sensor for 10 minutes. 500 μL of PBS was then flowed over the gold surface to remove poorly adsorbed EMT003 fusion protein. All measurements were performed at room temperature.

[0290] Two different types of antibodies were conjugated to EMT003 through multiple SPR channels. First, 10 μg / mL of anti-spike antibodies were flowed through channels B, C, and D. As a negative control, 10 μg / mL of anti-TGFB was injected into channel A. Then, two washing steps with PBS and PBST were performed to remove excess low-absorption antibodies against EMT003. Finally, a blocking step with BSA was included to prevent potential nonspecific binding of spike proteins to the sensor surface during the titration step.

[0291] Titration with clinically relevant concentrations of SARS-CoV-2 spike protein consisted of four injections of increasing concentrations of 10, 50, 100, and 200 ng / mL. SPR real-time binding profiles are provided in Figure 12A. A shift in RU is evident for SARS-CoV-2 anti-spike antibodies (red, blue, and green lines) over anti-TGFB antibodies at concentrations of spike protein above 100 ng / mL.

[0292] Additional titrations with higher concentrations of SARS-CoV-2 spike protein were also performed, consisting of five injections of increasing concentrations of 300, 625, 1250, 2500, and 5000 ng / mL, and are shown in Figure 12B. The shift in RU is significantly different between the SARS-CoV-2 anti-spike antibody and the anti-TGFB antibody negative control.

[0293] Conclusion: EMT003 conjugated with anti-spike antibody was able to detect recombinant spike antigen as low as 100ng / mL. EMT003 conjugated with anti-spike antibody can detect higher concentrations of recombinant spike protein in a linear and specific manner. The test is also specific, since EMT003 conjugated with anti-TGFB did not detect spike protein as expected for the negative control.

[0294] Example 12 Production and purification of streptavidin fusion proteins in Escherichia coli:

[0295] The protein sequences of the fusion proteins containing the gold-binding peptides in Table 6 below and fused to a linker and streptavidin were converted into streptavidin fusion proteins in the E. coli pET-30a(+) expression vector using the same cloning and purification strategy described in Example 1.

[0296] [Table 7]

[0297] As shown in Figure 13, gels A) and B) show the expression and purity of gold-conjugated streptavidin fusion proteins on a Coomassie stained SDS-PAGE gel. 2 μg of BSA was added to lane 1 of each gel A) and B). Gel A) shows the intact gold-conjugated streptavidin fusion protein EMT027, and gel B) shows the intact gold-conjugated streptavidin fusion protein EMT028.

[0298] Example 13 Lateral flow assay application of streptavidin fusion proteins: Gold-conjugated streptavidin fusion proteins EMT027 and EMT028 were conjugated to gold nanoparticles according to the method outlined in Example 5. Both gold-conjugated streptavidin fusion proteins were successfully conjugated to gold nanoparticles over a range of pH.

[0299] Then, the immobilization of biotinylated detection antibodies to gold nanoparticles coated with gold-conjugated streptavidin fusion protein and their antigen-binding ability were tested using a lateral flow assay. In this assay, antigens (rabbit IgG antibodies) were dotted directly on the strip membrane. Biotinylated detection antibodies (anti-rabbit IgG) were loaded onto the streptavidin fusion proteins (EMT027 and EMT028) immobilized on gold nanoparticles and then flowed up the membrane. As shown in Figure 14, the immobilized antigens on the strip can be detected by both EMT027 and EMT028-based conjugates (i.e., gold nanoparticle-streptavidin fusion protein-biotin conjugate detection antibody complexes) in the lateral flow assay. Specifically, 0.5 μg of rabbit antigen (rabbit IgG antibodies) was dotted on the membrane. Biotinylated or non-biotinylated anti-rabbit IgG was then loaded with either streptavidin fusion proteins (EMT027 and EMT028) immobilized on gold nanoparticles at various pHs for each fusion. Figure 14 shows three strips at each pH, ​​one with no anti-rabbit IgG loaded (left strip), one with biotinylated anti-rabbit IgG and streptavidin fusion (middle strip), and one with non-biotinylated anti-rabbit IgG and streptavidin fusion (right strip), demonstrating the specificity of anti-rabbit IgG specifically binding to the antigen when loaded and conjugated to the fusion protein (EMT027 or EMT028).

[0300] The nucleocapsid antigen binding ability of EMT028-based gold nanoparticle conjugates 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. EMT028-based gold nanoparticle conjugates were then mixed with nucleocapsid antigen and flowed up the membrane. As shown in Figure 15, EMT028-based gold nanoparticle conjugates loaded with biotin detection antibody (anti-nucleocapsid) successfully detected nucleocapsid antigen in the dotted sandwich lateral flow assay. Two different capture antibodies were evaluated and showed comparable results.

[0301] 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 Figure 16, the EMT028-based conjugate bound to nucleocapsid antibody successfully detected nucleocapsid antigen but not spike antigen in a striped sandwich lateral flow assay. No non-specific binding to 1ug / ml spike protein was observed, but a clear signal was obtained in the sample with nucleocapsid antigen. No non-specific binding was observed in the negative control sample. These results together indicate the specificity of the assay.

[0302] The detection of nucleocapsid antigen at 1 ng / ml and 5 ng / ml in artificial saliva containing mucin by EMT028 conjugate was also evaluated. In this assay, a sample volume of 60 μL was added to the lateral flow strip. As shown in FIG. 17, bands were clearly visible in both samples upon completion of the assay. These results indicate that EMT028 conjugate bound to nucleocapsid antibody in a striped sandwich lateral flow assay successfully detects nucleocapsid antigen in artificial saliva.

[0303] Example 14 Screening for nucleocapsid antibodies using EMT028 / biotin-nucleocapsid with SPR:

[0304] This study was conducted to evaluate the streptavidin fusion protein EMT028 conjugated with SARS-CoV-2 biotinylated nucleocapsid protein for antibody detection as an analyte using SPR.

[0305] First, EMT028 was diluted to 10 μg / mL. The clean gold-coated sensor was then loaded into the flow module in the SPR instrument. 500 μL of distilled water and 500 μL of PBS were flowed briefly over the sensor to establish a baseline signal. The fusion protein EMT028 was then flowed over the sensor for 10 min. PBS and PBS-Tween (0.005%) were then flowed over the gold surface to remove poorly adsorbed EMT028 fusion protein.

[0306] As the second layer of the system, biotinylated nucleocapsid protein was bound to EMT028. Then, one washing step with PBST was performed to remove excess 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 into channel A, while anti-spike antibody was injected into channel B (as a negative control). See Figure 18.

[0307] Interaction of anti-nucleocapsid MM08 antibody with biotinylated nucleocapsid protein showed a significant increase in signal shift. This signal remained constant after two PBST rinses, suggesting a strong and stable binding. No signal shift was observed when anti-spike was flowed over EMT028 / biotin-nucleocapsid. No major signal shift was observed for interaction of anti-spike 298 with biotinylated nucleocapsid protein.

[0308] Conclusion: EMT028 conjugated with biotinylated nucleocapsid protein was able to detect anti-nucleocapsid MM08 antibody at a concentration of 10 μg / mL, with no detectable binding to non-nucleocapsid antibodies, demonstrating a highly sensitive and specific detection system.

[0309] Example 15 Generation and purification of gold-binding fragments and bispecific immunoglobulin A fragments and bispecific antibody fragments. Bispecific antibodies and antibody fusion fragments are produced as known in the art. Specifically, genes of different antibodies or antibody fragments were cloned and transfected into Expi-CHO cells (Thermofisher) and then purified by AKTA Explorer protein purification system.

[0310] We clone, express, and purify bispecific Immunoglobulin A dimers, where one antibody monomer of the fused Immunoglobulin A dimer has high affinity for gold and the other antibody monomer has high affinity for the SARS-CoV-2 spike protein.

[0311] Surface plasmon resonance (SPR), an optoelectronic biosensing technique, is chosen to evaluate the binding kinetics of bispecific immunoglobulin A fusions to gold surfaces. First, the immobilization of bispecific immunoglobulin A fusions and two controls (ISBP-free fusion protein and buffer only) is evaluated. Zero or minimal binding is observed for these controls. However, the bispecific immunoglobulin A fusions show a 10-fold increase in resonance units (RU) during the immobilization phase compared to the ISBP-free control version. After it is established that the bispecific immunoglobulin A fusions are bound to the gold surface, a dilution series of spiked antigens is performed over the following concentrations in two experiments: 1.5625 nM (x2), 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM. Raw data is analyzed using Biacore™ 8K Evaluation Software version 1.1. SARS-CoV-2 spike antibodies are shown to bind bispecific immunoglobulin A fusions with a KD of 1–2E-10 M.

[0312] Example 16 In another example, a bispecific antibody fragment fusion with a gold-binding VH domain and an scFv specific for the SARS-CoV-2 spike protein is cloned, expressed, and purified using various methods known in the art. In a specific example, the fusion is cloned into a phagemid or other known cloning vector. The fusion containing a 6X His tag is cloned into an expression vector and transformed into a BL21(DE3) competent cell line and expression system. The transformation is performed under conditions in which heat shock is performed in ice → 42°C x 90 seconds → in ice. 750 μL of LB medium is added to the BL21 solution transformed by heat shock, and the whole is cultured at 37°C for 1 hour with shaking. Then, it is centrifuged at 6,000 rpm x 5 minutes, and 650 μL of the culture supernatant is discarded. The remaining culture supernatant and the cell fraction as a precipitate are stirred and plated on an LB / amp. plate, and the whole is left to stand overnight at 37°C.

[0313] Primary culture and expression

[0314] When it is confirmed that a clone has the desired fusion protein, the preculture solution containing the clone is subcultured in 750 mL of 2xYT medium, and the culture is continued at 28°C. 600 When the β-actin concentration exceeds 0.8, IPTG is added to a final concentration of 1 mM and the culture is carried out at 28° C. overnight.

[0315] purification:

[0316] The fusion protein is purified from the insoluble particulate fraction through the following steps:

[0317] (i) Collection of insoluble granules

[0318] The culture is centrifuged at 6,000 rpm for 30 minutes to obtain a precipitate as a bacterial fraction. The obtained material is suspended in a Tris solution (20 mM Tris / 500 mM NaCl) in ice. The obtained suspension is then homogenized in a French press to obtain a homogenized solution. The homogenized solution is then centrifuged at 12,000 rpm for 15 minutes to remove the supernatant and obtain a precipitate as an insoluble granular fraction containing inclusion bodies.

[0319] The insoluble fraction is then soaked overnight in 10 mL of 6 M guanidine hydrochloride / Tris solution, and centrifuged at 12,000 rpm for 10 minutes to obtain the supernatant as a solubilized solution.

[0320] (ii) Metal chelate column

[0321] A Ni column is used as the metal chelating column carrier. Column conditioning, sample loading, and washing steps are performed at room temperature (20° C.). Elution of the target His-tagged fusion protein is performed in 60 mM imidazole / Tris solution.

[0322] (iii) Refolding

[0323] The samples containing the fusion proteins are refolded using dialysis, immersed in a 6M guanidine hydrochloride / Tris solution and dialyzed for 6 hours with gentle agitation. The concentration of the external guanidine hydrochloride solution is slowly reduced stepwise over time into PBS buffer, in this case the fusion with the gold-binding VH domain and the scFv specific for the SARS-CoV-2 spike protein is properly refolded. Surface plasmon resonance (SPR), an optoelectronic biosensing technique, is chosen to evaluate the binding kinetics of the bispecific antibody fragments to the 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. However, the bispecific antibody fragment fusion shows a 10-fold increase in resonance units (RU) during the immobilization step compared to the ISBP-free control version. After the bispecific antibody fragment fusion is demonstrated to bind to the gold surface, a dilution series of spike antigen is performed across the following concentrations in two experiments: 1.5625 nM (x2), 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. Raw data is analyzed using Biacore™ 8K Evaluation Software version 1.1. The SARS-CoV-2 spike antibody is shown to bind to the bispecific antibody fragment fusion with a KD of 1-2E-10 M.

[0324] Example 17 Binding analysis of synthetic binding proteins to streptavidin and sensor-fused silica, polystyrene, and cellulose using QCM-D:

[0325] The protein sequences of the fusion proteins containing the cellulose, polystyrene or silica binding peptides in Table 7 below and fused to a linker and streptavidin were converted into fusion proteins in the E. coli pET-30a(+) expression vector using the same cloning and purification strategy described in Example 1.

[0326] [Table 8]

[0327] FIG. 21 shows Coomassie stained SDS-PAGE gels demonstrating the expression and purity of cellulose-bound streptavidin fusion proteins (FIGS. 21A-B), polystyrene-bound streptavidin fusion proteins (FIGS. 21C-D), and silica-bound streptavidin fusion proteins (FIG. 21E) of certain fusion proteins listed in Table 7.

[0328] For analyte detection, the fusion proteins in Table 7 were loaded onto respective silica, polystyrene, or cellulose sensors using a quartz crystal microbalance with dissipation monitoring (QCM-D) as the target surface, as shown in Table 7. All fusion proteins in Table 7 were diluted to a concentration of 25 μg / ml in 1x PBS solution in type 1 water.

[0329] The same PBS solution was used to dilute BSA to 100 μg / mL. All biotinylated antibodies for binding to streptavidin and their respective antigens for detection were diluted to a concentration of 25 μg / mL in 1x PBS solution in type 1 water. This includes troponin (antigen), anti-troponin antibody, and biotinylated troponin antibody.

[0330] Each QCM sensor was primed with PBS for approximately 3 hours. Then, each sensor was washed with fresh PBS for 5 minutes. Each fusion peptide diluted in PBS solution was loaded onto each sensor with the indicated inorganic surface for 1 hour. After the fusion peptide was absorbed onto the surface, the sensor was washed with PBS for 30 minutes, followed by BSA solution for 30 minutes, followed by PBS for 30 minutes. Next, biotinylated troponin antibody was loaded onto the surface for 40 minutes, followed by PBS for 30 minutes. Troponin antigen was then added for 15 minutes, followed by washing with PBS for another 30 minutes.

[0331] Tables 8 and 9 below summarize the modeled mass and thickness values ​​for each step of these QCM sensor experiments. The sensorgrams are shown in Figures 22A-E.

[0332] [Table 9]

[0333] [Table 10]

[0334] Figures 22A-E show the change in absorption of all fusions in Table 7 under this protocol. Specifically, Figures 22A and B show the absorption upon detection of nanometer-thick GL008 and GL009, respectively, on polystyrene surfaces. Notably, GL008 and GL009 polystyrene-bound fusion proteins showed different adsorption. After rinsing with PBS, GL009 showed a final adsorption of 5.9 nm, while GL008 showed 3.2 nm. Initially, the adsorption of GL008 was similar to GL009 for at least 5 nm of protein adsorption, after which a sudden desorption occurred during the protein adsorption step.

[0335] In any case, after fusion protein binding, there was minimal absorption of the BSA blocking agent, but substantial absorption of the biotinylated troponin antibody, indicating selective binding to streptavidin. Detection of binding to the antigen of interest (troponin) is also detected with both.

[0336] Figures 22C and D show the absorption upon detection of nanometer-thick EMT032 and EMT033, respectively, on a cellulose surface. Both cellulose-binding fusion proteins were found to bind to the cellulose sensor surface. Only a small fraction was washed off during the subsequent washing steps. Subsequent blocking and washing steps with BSA did not result in significant changes in layer thickness or mass. Biotinylated troponin antibody was substantially absorbed, indicating selective binding to streptavidin. However, these figures show that the troponin antigen was minimally adsorbed compared to other surfaces or fusion peptides.

[0337] FIG. 22E shows the absorption upon detection of nanometer-thick EMT029 on the silica surface, respectively. Despite significantly less fusion protein adsorption compared to the other sensors, based on the observed slope of the raw data frequencies, it is likely that there would have been additional EMT029 fusion protein adsorption if the flow time had been extended beyond 1 hour in this step (i.e., the step had not yet reached full equilibrium). Absorption of biotinylated troponin antibody was also shown, showing selective binding to streptavidin, especially when compared to the PBS blocker. Finally, there was substantial absorption when troponin antigen was added.

[0338] Example 18 Bispecific scFv antibodies

[0339] In another example, a bispecific antibody fragment fusion with a gold-binding VH domain and an scFv specific for troponin was cloned, expressed and purified using various methods known in the art. The scFv troponin fusion (GL007) comprises the sequence shown in Table 10 below and in Figure 23.

[0340] [Table 11-1] [Table 11-2]

[0341] Specifically, FIG. 23 shows a 6X His tag fused to a TEV cleavage site, followed by a VH domain, a gold binding motif, followed by linker 1, followed by a VH anti-troponin domain, followed by a linker, followed by a VL anti-troponin domain.

[0342] This fusion was cloned into expression vectors and systems well known in the art. The fusion protein was purified from the insoluble particulate fraction by the following steps:

[0343] (i) Collection of insoluble granules

[0344] The culture was centrifuged at 6,000 rpm for 30 minutes to obtain a precipitate as a bacterial fraction. The obtained material was suspended in a Tris solution (20 mM Tris / 500 mM NaCl) in ice. The resulting suspension was then homogenized in a French press to obtain a homogenized solution. The homogenized solution was then centrifuged at 12,000 rpm for 15 minutes to remove the supernatant and obtain a precipitate as an insoluble granular fraction containing inclusion bodies.

[0345] The insoluble fraction was then immersed overnight in 10 mL of 6 M guanidine hydrochloride / Tris solution, and the resultant was centrifuged at 12,000 rpm for 10 minutes to obtain the supernatant as a solubilized solution.

[0346] (ii) Metal chelate column

[0347] A Ni column was used as a metal chelating column carrier. Column conditioning, sample loading, and washing steps were performed at room temperature (20°C). Elution of the target His-tagged fusion protein was performed in 60 mM imidazole / Tris solution.

[0348] (iii) Refolding

[0349] The samples containing the fusion proteins were refolded using dialysis, immersed in 6 M guanidine hydrochloride / Tris solution and dialyzed for 6 h with gentle agitation. The concentration of the external guanidine hydrochloride solution was slowly reduced stepwise over time into PBS buffer, in which case the fusions with the gold-binding VH domain and the troponin-specific scFv were properly refolded.

[0350] Figure 24 shows a Coomassie stained SDS-PAGE gel showing the expression and purity of the bispecific antibody SEQ ID NO: 29. The scFv troponin fusion SEQ ID NO: 29 was then loaded onto a gold target surface as shown using a quartz crystal microbalance with dissipation monitoring (QCM-D).

[0351] The fusion protein and troponin antigen were diluted to a concentration of 25 μg / ml in 1×PBS solution in water type 1. BSA was diluted to 100 μg / mL using the same PBS solution.

[0352] The QCM sensors were then primed with PBS for approximately 1 hour and then washed with fresh PBS for 5 minutes. The scFv troponin fusions diluted in PBS solution were loaded onto two gold surface sensors for 1 hour. After allowing the fusion peptide to absorb onto the surface, the sensors were washed with PBS for 30 minutes, followed by BSA solution for 30 minutes, followed by PBS for 30 minutes. Troponin antigen or spiked antigen control was then added to the respective sensors for 15 minutes, followed by washing with PBS for another 30 minutes. Figure 25A-B shows the change in absorption under this protocol and Table 11 shows the change in mass and thickness values.

[0353] [Table 12]

[0354] After adsorption of GL007 to the gold sensor, negligible thickness changes are detected during the subsequent PBS rinsing and BSA blocking steps. Although there is some initial adsorption of troponin antigen to the sensor, minimal final adsorption of troponin antigen was observed after rinsing with PBS.

[0355] Example 19 Lateral Flow Assay Streptavidin Fusion Protein:

[0356] GL011 was first produced by cloning and amplifying in a recombinant baculovirus Sf9 insect cell system. The gene for GL011 was inserted into plasmid DNA as known in the art using a QIAGEN miniprep DNA purification kit. Sf9 cells were also seeded in insect cell medium in 6-well tissue culture plates and allowed to attach.

[0357] 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 / antibiotic-free medium were mixed and incubated at room temperature for 15 minutes. The medium from the cells was replaced with 2 milliliters of FBS / antibiotic-free medium. The wash medium was removed and the transfection mix complex was overlaid onto the washed cells at 60 rpm and shaken at 27°C for 4 hours. Once the recombinant baculovirus was transfected with the GL011 gene, the baculovirus was amplified with Sf9 cells in a T75 flask using the SignalChem Pharmaceutical Sf9 Amplification System.

[0358] To express the recombinant GL011 protein, 3x10 cells were cultured in 300 ml of Excell-400 medium (JHR Biosciences). 8 Sf9 cells are mixed with approximately 5 MOI of baculovirus in a spinner flask and shaken at 80 RPM for 72 hours at 27° C. The Sf9 cells are then harvested by centrifugation of the medium and removal of the supernatant. The pellet is lysed and purified along with the His tag of the GL011 protein using the Talon Cobalt bead system.

[0359] Figure 26 shows the purity of GL011 His-tagged gold-bound streptavidin fusion protein on a Coomassie stained SDS-PAGE gel. The amino acid sequence of GL011 is confirmed by the following sequence: affinity tag (his tag)-streptavidin and linker (SEQ ID NO: 1) fusion with gold protein (98 amino acid gold protein).

[0360] [Table 13]

[0361] The gold-binding streptavidin fusion protein GL011 was then conjugated to gold nanoparticles according to the methods outlined in Example 5.

[0362] The immobilization of biotinylated detection antibodies to gold nanoparticles coated with gold-conjugated streptavidin fusion protein and their antigen-binding ability were then tested using a lateral flow assay, in which antigen (SARS-CoV-2 nucleocapsid antigen) at various antigen concentrations was dotted directly onto a strip membrane. Specifically, SARS-CoV-2 nucleocapsid antigen was diluted in pooled human saliva at 100ng / mL, 10ng / mL, and 2ng / mL and then dotted individually onto the lateral flow assay membrane.

[0363] Biotinylated detection antibody (SARS-CoV-2 nucleocapsid antibody) was loaded onto the streptavidin fusion protein GL011 immobilized on gold nanoparticles and then flowed up the membrane. As shown in Figure 27, with immobilized nucleocapsid antigen, the strip can be detected by GL011 conjugate (i.e., gold nanoparticle-streptavidin fusion protein-biotin conjugate detection antibody complex) in a lateral flow assay, and is specifically detected at a low concentration of 2ng / mL and as shown by the blank control without nucleocapsid antigen. These results indicate that the EMT011 conjugate bound to the nucleocapsid antibody in the striped lateral flow assay successfully detects nucleocapsid antigen in artificial saliva.

[0364] In another example using GL011, the gold-bound streptavidin fusion protein GL011 was again conjugated to gold nanoparticles according to the method outlined in Example 5. A biotinylated detection antibody (SARS-CoV-2 nucleocapsid antibody) was then loaded onto the streptavidin fusion protein G011 immobilized on the gold nanoparticles.

[0365] This assay, a rapid lateral flow assay, detected SARS-CoV-2 nucleocapsid antigen from human anterior nasal swab samples. Specifically, a control line of polyclonal anti-IgG antibodies was placed on the surface of the test strip. Additionally, a test line containing anti-covid antibodies was placed on the surface of the test strip.

[0366] To prepare for the test, the nasal swab sample was added to a buffer solution in a sample collection tube. This buffer solution contains a biotinylated detection antibody (SARS-CoV-2 nucleocapsid antibody) loaded onto a conjugated GL011 fusion protein. Then, after incubation, the sample was added to the sample well of the test strip to allow lateral flow of the sample on the test strip. After complete lateral flow, the test detected SARS-CoV-2 nucleocapsid antigen in human samples with at least 84% sensitivity and 99% specificity.

[0367] Example 20 Lateral flow assay:

[0368] An exemplary assay utilizing (nitro)cellulose-bound streptavidin fusion proteins (e.g., EMT032-EMT033) and a dual affinity probe for detecting or quantifying analyte levels (SARS-CoV-2 nucleocapsid protein) in a test sample is described. The assay is manufactured into a diagnostic kit using the dual affinity probe according to the method described above. As shown in FIG. 29, the kit has a pretreated sample pad section for collecting and reacting a biological sample from a subject, a nitrocellulose membrane section where the detection complex is formed on the test line, and an absorbent pad at the end of the strip for collecting the flow-through fraction. In this particular example, the pretreated sample pad contains an analyte composition comprising lyophilized beads that contain two components: 1) encapsulated physisorbed antibody-gold nanoparticles (the antibody is a capture element antibody that specifically binds to the analyte SARS-CoV-2 nucleocapsid protein), and 2) free biotinylated antibodies that also specifically bind to a different epitope of the analyte SARS-CoV-2 nucleocapsid protein. The sample pad is treated with lyophilized beads immediately prior to use of the kit, or alternatively, the beads are added during manufacture of the kit.

[0369] A test sample having the analyte of interest is collected from a subject using various collection methods (e.g., collecting a spat sample in a tube, collecting a nasal swap using an assay strip, etc.) and the collected test sample is introduced into a reagent solution. The reagent solution containing the test sample is then applied directly onto the pre-treated sample pad.

[0370] The sample containing the analyte flows laterally to and then contacts the lyophilized beads, which dissolve in the sample reagent and form a sandwich of biotinylated analyte-binding antibodies bound to the analyte, which also bind to antibody-conjugated gold nanoparticles, with the analyte SARS-CoV-2 nucleocapsid protein sandwiched between both antibodies. These sandwich complexes form within the sample pad before sample flow begins, resulting in more controllable stoichiometry and reduced margin of error. The sandwich complexes then flow laterally over the surface of the nitrocellulose membrane, where they are captured on the test line by forming a complex (i.e., detection conjugate) with an immobilized streptavidin fusion protein. The presence of the detection conjugate captured at the test line is observed with the naked eye to determine the presence and / or amount of analyte in the test sample.

[0371] Example 21 Competitive sandwich lateral flow assay:

[0372] With reference to FIG. 29, this assay is performed to assess the presence or amount of an analyte of interest (SARS-CoV-2 nucleocapsid protein) in a test sample. First, similar to the sample collection protocol described in Example 20, a test sample is collected from a subject, mixed with a reagent, and applied onto a pre-treated sample pad containing lyophilized beads containing a dual affinity probe. The dual affinity probe contains biotinylated gold nanoparticles coated with an antigen (recombinant SARS-CoV-2 nucleocapsid protein, not derived from the sample) engineered to be immobilized to gold via a gold binding motif. The antigen is also bound to a capture element biotinylated antibody designed to specifically and competitively bind to that antigen and to the SARS-CoV-2 nucleocapsid protein derived from that sample. When the sample is added to the pre-treated sample and flows laterally to the lyophilized beads, the lyophilized beads dissolve into the sample reagent, allowing this competitive binding reaction to occur (i.e., the analyte in the sample and the gold-immobilized antigen derived from the beads competitively bind to the anti-analyte antibody). The lyophilized beads flow down to the surface, making the stoichiometry more controlled and reducing the margin of error. The gold nanoparticle-coated antigen-bound biotinylated antibody complex, along with the analyte-bound biotinylated antibody, then flows laterally over the surface of the nitrocellulose membrane, where it is captured on the test line by forming a complex with the immobilized streptavidin fusion protein. More analyte in the sample will result in more antibody displacement from the gold nanoparticles, and therefore less gold capture complex will be observed at the test line if the analyte SARS-CoV-2 nucleocapsid protein is present in the sample. The amount of analyte in the sample is observed with the naked eye by determining the intensity of the signal.

[0373] Example 22 Multi-bead sandwich lateral flow assay:

[0374] This assay kit uses two different analyte compositions of lyophilized beads for pretreatment of the sample pad of the assay strip. An exemplary embodiment is shown in Figure 30A-B. As shown in Figure 30A, a sample containing the analyte of interest (SARS-CoV-2 spike protein) is collected with a reagent and applied onto the pretreated sample pad. The sample flows laterally down the sample pad, where the sample reagent dissolves the first analyte composition of the lyophilized beads. The first analyte composition of the lyophilized beads contains gold nanoparticles coated with an immobilized antigen (recombinant SARS-CoV-2 spike protein not derived from the sample) fused to a gold binding protein. When the analyte and antigen-bound gold nanoparticles from the sample mix in the sample reagent, the mixture is further contacted with a second analyte composition of lyophilized beads containing a biotinylated capture element antibody that specifically binds to the antigen and the analyte. Upon contact, the beads of the second analyte composition dissolve and release the biotinylated antibody, where the analyte gold beads and the analyte competitively bind to the biotinylated capture element antibody. Excess analyte in the mixture outcompetes the gold-immobilized antigen for antibody binding.

[0375] This complex formation occurs in the sample pad before the lateral flow begins, resulting in more controlled stoichiometry and reduced margin of error. The gold nanoparticle-antigen-bound biotinylated antibody complex then flows laterally over the surface of the nitrocellulose membrane along with the analyte-bound biotinylated antibody, where it is captured on the test line by forming a complex with the immobilized streptavidin fusion protein. A positive control can also be run with the sample by adding gold nanoparticle-conjugated chicken IgY antibody to the second group of lyophilized beads and immobilizing anti-chicken IgY antibody on the cellulose membrane on the control line.

[0376] The more analyte in the sample, the more antibody that competes with the antigen-bound gold nanoparticles will be captured, and therefore, less gold capture complex will be observed at the test line when the analyte is provided in the sample. The amount of analyte in the sample is observed with the naked eye by determining the intensity of the signal.

[0377] Example 23 Multi-bead sandwich lateral flow assay:

[0378] In another exemplary embodiment, the sample collection pad of the assay kit is pretreated with two different analyte compositions, each containing lyophilized beads. The first analyte composition of the beads contains an antigen (a recombinant SARS-CoV-2 spike protein not derived from the sample) fused to a gold binding protein coated with gold nanoparticles. The analyte is also bound to a non-biotinylated IgG mouse antibody that binds to the antigen and can also bind to the analyte. The second analyte composition contains lyophilized beads with i) a universal capture biotinylated anti-mouse IgG antibody and ii) a gold nanoparticle control coated with chicken IgY. As shown in Figures 31A-31B, the two analyte compositions are processed side-by-side on the pretreated sample pad. A sample containing the analyte (SARS-CoV-2 spike protein) is collected and added to the pretreated sample pad, where the sample reagent dissolves the first analyte composition of the lyophilized beads. When analytes from the sample compete with the gold-immobilized antigen for antibody binding, the second group of beads dissolves, releasing the secondary antibody and forming a biotinylated antigen-primary antibody-secondary antibody complex. The complex, together with the chicken IgY antibody and excess unbound antigen-coated gold nanoparticles, then flows laterally over the surface of the nitrocellulose membrane, where it is captured at the test line by forming a complex with the immobilized streptavidin fusion protein. Alternatively, the chicken IgY released from the second group of beads is captured on the control line by the immobilized anti-chicken IgY antibody (Figure 31B).

[0379] Example 24 Multi-bead sandwich lateral flow assay:

[0380] This is another exemplary assay kit for detecting the presence of an analyte in a sample. In this kit, as shown in FIG. 32A, a sample collection pad is treated with two analyte compositions: i) lyophilized beads having a mouse monoclonal IgG antibody (i.e., anti-analyte SARS-CoV-2 spike protein antibody) and ii) a complex of gold nanoparticles bound to a streptavidin fusion protein by a gold-binding protein-conjugated biotinylated anti-mouse IgG antibody. As described in Example 1, a sample is collected from a subject using various collection methods, and the collected sample is placed in a reagent and applied onto the sample pad. Upon dissolving the first bead of the first analyte composition in the sample reagent, the analyte SARS-CoV-2 spike protein in the sample can be recognized by the mouse monoclonal IgG antibody and then interact with the secondary mouse antibody in the second analyte composition containing a complex with a streptavidin fusion protein and gold nanoparticles (FIG. 32B-C). A positive assay control is detected at the control line when excess of the primary mouse antibody that is unbound to the analyte forms a complex with the immobilized rabbit polyclonal anti-mouse IgG antibody. The presence of the detection conjugate captured at the test line is observed with the naked eye to determine the presence and / or amount of analyte in the test sample.

[0381] Example 25 Sandwich lateral flow assay application of gold binder protein G complex:

[0382] Referring to FIG. 33, another exemplary sandwich detection complex is described. In this assay, the sample collection pad contains a dual affinity probe with a gold detection conjugate formed by indirectly binding an analyte capture element detection IgG antibody onto a gold nanoparticle using a protein G-gold binding motif fusion protein. The protein G-gold binding motif fusion protein is fused to a gold particle, which allows protein G to bind to an IgG antibody specific for the analyte (SARS-CoV-2 spike protein). On the test line of the assay, a nitrocellulose binder streptavidin fusion protein is bound to a biotinylated IgG capture antibody. This biotinylated IgG capture antibody is also specific for the analyte, but at a different epitope than the IgG antibody of the dual affinity probe. Samples collected from a subject using various collection methods as described in Example 1 are added to the sample pad, where the analyte (or antigen) in the sample reagent interacts with the gold detection conjugate to form a complex. The analyte complex containing the SARS-CoV-2 spike protein then flows laterally through the nitrocellulose membrane and is captured by the streptavidin fusion protein-conjugated antibody. The presence of the captured detection conjugate at the test line is observed with the naked eye to determine the presence and / or amount of analyte in the test sample.

[0383] Example 26 Sandwich Lateral Flow Assay:

[0384] Another exemplary assay using biotinylated sandwich anti-analyte antibodies and anti-biotin antibodies immobilized on a nitrocellulose membrane is described. As shown in Figures 34-35, the assay kit has a pretreated sample pad portion for collecting and reacting a sample from a subject, a nitrocellulose membrane portion where a sandwich complex is formed on the test line, and an absorbent pad at the end of the strip for collecting excess flow-through. In this particular example, the pretreated sample pad contains an analyte composition that includes two components: i) gold surface particles bound to a physisorbed analyte capture antibody that includes a gold-binding peptide, and ii) a dual affinity probe that includes a tagged sandwich antibody that is also specific for binding the analyte SARS-CoV-2 spike protein. Both antibodies are designed to bind different epitopes of the analyte SARS-CoV-2 spike protein, thereby allowing a complete sandwich if the analyte is present.

[0385] A test sample with the analyte of interest is collected from a subject using various collection methods (e.g., collecting a spat sample in a tube, collecting a nasal swap using an assay strip, etc.), and the collected test sample is applied directly to a pre-treated sample pad or pre-mixed with kit reagents in a separate tube before application. When the sample reagents contact the lyophilized beads, the beads dissolve, allowing the analyte SARS-CoV-2 spike protein in the sample to form a complete antibody sandwich complex. The sandwich complex formed at the sample pad then begins to flow laterally on the surface of the membrane. At the test line, the sandwich complex is captured by binding the tagged antibody with an anti-biotin (Figure 34) or anti-His (Figure 35) antibody via the biotin or His tag on the tagged sandwich antibody. The presence of the sandwich detection conjugate captured at the test line is observed with the naked eye to determine the presence and / or amount of analyte in the test sample.

[0386] Example 27 Antibody-based sandwich lateral flow assay: Another exemplary assay utilizes antibodies grown in two different species (e.g., two different species, such as mouse and chicken, or two different IgG antibody isotypes (e.g., IgG1 and IgG2)) to detect the analyte SARS-CoV-2 spike protein in the sample. In a particular embodiment, a biological sample from a subject is placed on a pretreated sample pad, which holds an analyte composition containing a dual affinity probe including a physisorbed secondary antibody with a gold-binding peptide that binds to gold nanoparticles. This secondary antibody is used to bind to a single species, such as IgG1. The anti-IgG1 antibody is the primary anti-analyte antibody and is bound to an IgG1 antibody that can specifically bind to the analyte SARS-CoV-2 spike protein. The analyte composition includes a second binding moiety that is an antibody of a different species than the primary anti-analyte antibody (in this case, an IgG2 antibody) that can specifically bind to the analyte SARS-CoV-2 spike protein at a different epitope than the IgG1 antibody. The sample containing the analyte is added to the pretreated sample and then flows laterally to the analyte composition on the pretreated sample pad. There are two different species of anti-analyte antibodies that bind to the analyte sandwich and form a complete antibody sandwich. As shown in Figures 36-37, the analyte composition can include a dual affinity probe with or without lyophilized beads and can be added to the pretreated sample pad in multiple ways, including using a different surface or the same surface as the surface for the sample. As the sample flows to the analyte composition, a sandwich complex forms due to the presence of the analyte and then flows laterally down the nitrocellulose membrane. In this case, the complex is captured by the immobilized secondary antibodies (anti-IgG1 and anti-IgG2 antibodies) on the nitrocellulose membrane. The sandwich containing the analyte is captured and detected on the test line, whereas the unbound (no analyte) is detected on the control line (anti-IgG1). The presence of the sandwich detection conjugate captured at the test line can be observed with the naked eye to determine the presence and / or amount of analyte in the test sample.

[0387] Example 28 Lateral flow assay:

[0388] Exemplary assays are described that utilize cellulose-bound streptavidin fusion proteins (e.g., EMT033, GL018, GL019, and GL020) to detect or quantitate analyte levels (SARS-CoV-2 nucleocapsid protein) in a test sample.

[0389] For immobilization of biotinylated SARS-CoV-2 anti-nucleocapsid antigen capture antibody, EMT033 cellulose-bound streptavidin fusion protein was striped onto nitrocellulose and cellulose strips.

[0390] Two specific assays were performed: a wet assay and a dry assay.

[0391] In the wet assay, gold beads were conjugated with two separate SARS-CoV-2 anti-nucleocapsid detection antibodies and mixed with biotinylated anti-nucleocapsid antibody, buffer, and nucleocapsid antigen. Three different concentrations of nucleocapsid antigen were tested: 0 ng / mL, 37.5 ng / mL, and 300 ng / mL.

[0392] FIG. 39 shows the results of a wet assay where EMT033 cellulose-bound streptavidin fusion protein was striped onto nitrocellulose with nucleocapsid antigen at all three concentrations, showing a much stronger signal at the 300 ng / mL concentration.

[0393] FIG. 40 shows the results of a wet assay where EMT033 cellulose-bound streptavidin fusion protein was striped onto cellulose (Whatman 43 paper) with nucleocapsid antigen at all three concentrations, showing a much stronger signal at the 300 ng / mL concentration.

[0394] In the dry assay, gold beads were conjugated with two separate SARS-CoV-2 anti-nucleocapsid detection antibodies and then added to a conjugate pad and allowed to dry. A biotinylated SARS-CoV-2 anti-nucleocapsid antibody was then added to a separate but adjacent glass fiber pad and allowed to dry.

[0395] Next, a dry assay was performed using running buffer containing both SARS-CoV-2 nucleocapsid antigen and various concentrations of UV-inactivated whole SARS-CoV-2 virus. Figure 41 shows the results of the dry assay when EMT033 cellulose-bound streptavidin fusion protein was striped onto cellulose (Whatman 43 paper), demonstrating that this cellulose-based dry lateral flow assay was able to detect 10% of the virus in the low 10% range. 3 It has been shown to detect whole SARS-CoV-2 virus down to pfu / mL of virus.

[0396] The comparative dry assay utilized the same dry assay as above, but compared EMT033 cellulose-bound streptavidin fusion protein stripped onto a cellulose strip to immobilize the biotinylated SARS-CoV-2 anti-nucleocapsid antigen capture antibody, or biotinylated SARS-CoV-2 anti-nucleocapsid antigen capture antibody stripped by physical adsorption to the cellulose strip (i.e., without any EMT033 cellulose-bound streptavidin fusion protein). Figure 42 shows the results using a fixed concentration of SARS-CoV-2 nucleocapsid antigen, demonstrating detection of SARS-CoV-2 nucleocapsid antigen both with the use of cellulose-bound streptavidin fusion protein or without the fusion protein.

[0397] Example 29 PDMS binding fusion protein to PDMS surface via QCM-D:

[0398] Dual affinity probes were made using affinity tag (his tag)-streptavidin linker fusions with various PDMS binding motifs, i.e. GL014 (SEQ ID NO: 56), GL015 (SEQ ID NO: 57), and GL016 (SEQ ID NO: 58) fusions. Assay conditions similar to Examples 1 and 6 above, but using a PDMS surface.

[0399] Specifically, all QCM-D analyses were performed on a 4-channel Qsense™ analyzer (Biolin Scientific, Gothenburg, Sweden) at 23°C. PDMS Qsense™ sensor chips were rinsed and primed with PBS for 1 hour. An additional PBS run was performed for 5 minutes. GL014 (SEQ ID NO:56), GL015 (SEQ ID NO:57), and GL016 (SEQ ID NO:58) fusions were flowed on the respective PDMS sensor chips for 1 hour, followed by a 10 mM PBS wash step for 30 minutes, followed by a BSA blocking step for 30 minutes, and another PBS wash step for 30 minutes. Binding of GL014, GL015, and GL016 fusions to the surface was effected using Qsense™ Dfind™ analysis software. To test as dual affinity probes, biotinylated N antigen antibodies were then flowed on the respective fusions for 40 minutes, followed by a 10 mM PBS wash step for 30 minutes. SARS-CoV-2 nucleocapsid antigen was then flowed over each channel for 15 minutes, followed by a 10 mM PBS wash step for 30 minutes. Raw data was analyzed using the Kelvin-Voigt viscoelastic model with Qsense™ Dfind™ analysis software.

[0400] The thickness, deposited mass, elasticity, and viscosity of the layer obtained after each step were calculated and are summarized in Tables 13-15 for each fusion protein, respectively.

[0401] [Table 14]

[0402] [Table 15]

[0403] [Table 16]

[0404] Figure 43A-C show the absorbance change of each fusion moiety GL014, GL015, and GL016 under this protocol, respectively. All PDMS-bound fusion proteins were found to bind to the PDMS sensor surface. Only a small fraction of GL014 and GL016 was washed off during the subsequent washing steps, and a larger fraction of GL015 was washed off during the subsequent washing steps. There was no significant change in the thickness or mass of the layer during the subsequent BSA blocking or washing steps. As seen in Figure 43, a thick layer of biotinylated N antibody was adsorbed to the fusion protein and a similar amount of N antigen was adsorbed to the N antibody, indicating a sensitive and functional dual affinity probe using various PDMS fusion proteins on the QCM-D system.

[0405] While exemplary embodiments have been described above and illustrated in the accompanying drawings, it will be apparent to those skilled in the art that modifications may be made without departing from the present disclosure, and such modifications are considered as contemplated variations falling within the scope of the present disclosure.

[0406] All publications, patents, and patent applications, including any drawings and addenda, are incorporated by reference in their entirety into this specification for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or addendum was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

1. A biaffinity probe for detecting a sample in a sample, comprising a surface binding portion (SBM) and a capture element (CE), wherein the probe specifically binds to a surface material selected from the group consisting of gold, silica, silver, cellulose, plastic, polystyrene, and graphene.

2. The biaffinity probe according to claim 1, wherein the capture element (CE) is directly or indirectly connected to the SBM via one or more linkers (LI), and each LI is independently a single bond or an amino acid sequence.

3. The biaffinity probe according to claim 1 or 2, wherein the probe has the following formula (Ia) or formula (IIa): SBM-LI-CE(Ia) or CE-LI-SBM(IIa).

4. The aforementioned capture element (CE) i. Antibodies or their antigen-binding fragments, single-chain variable fragments (scFv) or Fab fragments, ii. antigen: A biaffinity immunoprobe according to any one of claims 1 to 3, comprising:

5. A biaffinity probe according to any one of claims 1 to 4, wherein LI is a single bond, or selected from one or more of the group consisting of a peptide or amino acid linker, an amino acid sequence containing protein G from Streptococcus, and an amino acid sequence containing streptavidin from Streptomyces.

6. The biaffinity probe according to any one of claims 1 to 5, wherein the SBM is selected from one or more of the group consisting of a binding peptide, a protein, an antibody having affinity for the surface material, an immunogenic fragment thereof, a single-chain variable fragment (scFv), and a Fab fragment.

7. The biaffinity probe according to claim 6, wherein the SBM is a conjugated peptide selected from the group consisting of any peptide sequences in Table 1 of this specification.

8. The biaffinity probe according to claim 1 or 2, wherein the biaffinity probe comprises a polypeptide having the following formula (IIIa) or (IIIb), or a polypeptide having the formula (IVa) or (IVb): SBM-LI-AL(IIIa) AL-LI-SBM (IIIb), ALB-LI-CE(IVa), or CE-LI-ALB(IVb) (wherein AL is the active linker and ALB is the active linker binder).

9. The biaffinity probe according to claim 8, wherein AL is an amino acid sequence containing protein G derived from Streptococcus, or an amino acid sequence containing streptavidin derived from Streptomyces.

10. The biaffinity probe according to any one of claims 1 to 9, wherein the CE is an antigen, an antibody or its antigen-binding fragment, scFv or Fab, an aptamer, MIP, or a manipulated protein.

11. The biaffinity probe according to claim 10, wherein the CE is an antigen, antibody, or an antigen-binding fragment thereof, the antigen, antibody, or an antigen-binding fragment thereof is conjugated with biotin, and the LI comprises an amino acid sequence containing streptavidin derived from Streptomyces.

12. The biaffinity probe according to claim 11, wherein the CE is an antibody or an antigen-binding fragment thereof, and the LI is an amino acid sequence containing protein G derived from Streptococcus.

13. A biaffinity probe according to any one of claims 1 to 10, wherein LI is a single bond, or a peptide or an amino acid linker.

14. The biaffinity probe according to claim 13, wherein the biaffinity probe is a single fusion protein.

15. The biaffinity probe according to claim 4, wherein the CE is a single-chain variable fragment from an antibody, and the SBM and the CE are fused as a bispecific antibody fragment.

16. The aforementioned SBM is V Ｈ The biaffinity probe according to claim 15, which is a single-chain variable fragment that is a gold-bonding motif.

17. A method for determining the presence of a sample in a test sample and / or for quantifying the sample, i. A biaffinity probe according to any one of claims 1 to 16, which includes a surface binding portion (SBM) and a sample-specific capture element (CE), is brought into contact with a test sample for a period of time and under conditions sufficient to bind the sample present in the test sample to the sample-specific capture element, thereby forming a complex containing the sample bound to the biaffinity probe. ii. Determining the presence and / or amount of the sample present in the complex or the complex, iii. The presence of the sample in the test sample is indicated by the presence of the sample in the composite or in the composite, and the amount of the sample in the test sample is indicated by the amount of the sample present in the test sample. iv. thereby determining the presence of the sample in the test sample and / or quantifying the sample. The aforementioned method.

18. A biaffinity probe for detecting a sample in a sample, i) Surface binding portions (SBMs) fused to or bonded to non-sample capturing elements (NACEs), ii) Surface particles fused to or bonded to NACE or trapping elements (CE) The biaffinity probe, including the above.

19. The biaffinity probe according to claim 18, wherein the NACE is one or more selected from the group consisting of a linker (LI), streptavidin, protein G, biotin, antigen protein or peptide, or an antibody or its conjugated fragment, the antibody is a non-fragmented antibody, a modified antibody, a single-chain variable fragment from an antibody, or a Fab fragment, and the CE is an antibody or its conjugated fragment selected from the group consisting of a non-fragmented antibody, a modified antibody, a camelid antibody, a single-chain variable fragment, a Fab fragment, and an antigen protein or peptide.

20. The biaffinity probe according to claim 18 or 19, wherein the surface particles bind to surface-bound peptides (SBPs), the SBPs fuse to or bind to non-sample-capturing elements (NACEs), the NACEs further fuse to or bind to CEs, and the biaffinity probe has the following formula I or formula II: (Surface particles)-SBP-NACE-CE (Formula Ic), or (Surface particles)-SBP-CE (Formula IIc).

21. The biaffinity probe according to any one of claims 18 to 20, wherein the NACE comprises one or more selected from the group consisting of a linker, streptavidin, protein G, biotin, biotinylated protein, antigen protein or peptide, and an antibody or its binding fragment, and the antibody is an unfragmented antibody, a modified antibody, a camelid antibody, a single-chain variable fragment, or a Fab fragment.

22. The biaffinity probe according to any one of claims 18 to 21, wherein the NACE comprises an antibody or its binding fragment that specifically binds to IgG, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgD, and IgE, an unfragmented antibody, a modified antibody, a camelid antibody, a single-chain variable fragment, or a Fab fragment.

23. The biaffinity probe according to any one of claims 18 to 21, wherein the NACE comprises protein G and / or streptavidin.

24. A diagnostic kit for detecting a sample in a sample, a) Surface and, b) A surface bonding portion (SBM) bonded to at least a part of the surface, c) Reagents for collecting the sample, d) A sample composition comprising at least one set of biaffinity probes according to claims 1 to 16 or 18 to 23, The diagnostic kit, including the above.

25. A biaffinity probe for detecting a sample in a sample, wherein the biaffinity probe is i) A surface binding moiety (SBM) which is a gold-binding protein (GBP) fused to or bound to a non-sample capturing element (NACE), or ii) Surface binding moieties (SBMs) which are gold-binding proteins (GBPs) fused to or bound to the capture element (CE), The biaffinity probe, including the above.

26. The biaffinity probe according to any one of claims 1 to 16, 18 to 23, or 25, wherein the probe specifically binds to cellulose, and the probe comprises a peptide sequence selected from the group consisting of SEQ ID NOs: 27 and 28.

27. The biaffinity probe according to any one of claims 1 to 16, 18 to 23, or 25, wherein the surface material is gold, and the probe comprises a peptide sequence selected from the group consisting of SEQ ID NOs: 20, 23, 24, and 34.

28. The biaffinity probe according to any one of claims 1 to 16, 18 to 23, or 25, wherein the surface material is silica and the probe contains the peptide sequence of SEQ ID NO:

26.

29. The biaffinity probe according to claim 1 or 2, wherein one or both of the CE and the SBM are antibodies, and the CE and the SBM are fused to form bispecific immunoglobulin A.

30. A diagnostic kit for detecting a sample in a sample, a) Surface and, b) A surface bonding portion (SBM) bonded to at least a part of the surface, c) Reagents for collecting the sample, d) A sample composition comprising at least one set of biaffinity probes according to claim 1 or 2, The diagnostic kit, including the above.

31. A diagnostic kit for detecting a sample in a sample, a) Cellulose or nitrocellulose surface and b) A surface bonding portion (SBM) including a cellulose or nitrocellulose bonding portion bonded to at least a portion of the cellulose or nitrocellulose surface, c) Reagents for collecting the sample, d) i) A biaffinity probe according to claim 1 or 2, comprising an antibody-bound gold particle, wherein the antibody is specific to binding to the sample and is bound to the gold particle by a gold-binding protein, and ii) a sample composition comprising a second binding partial antibody that is specific to the sample and has a binding tag, The diagnostic kit, including the above.

32. The diagnostic kit according to claim 30 or 31, wherein the biaffinity probe comprises a conjugated peptide selected from the group consisting of SBM or any peptide sequence in Table 1.

33. A method for determining the presence of a sample in a sample and / or quantifying the sample by testing a sample from a subject using any one of the diagnostic kits described in claims 30 to 32, a. Contacting the test sample with the reagent, b. Applying the test sample together with the reagent to the surface, thereby causing the test sample to flow laterally on the surface and bringing it into contact with the biaffinity probe composition on the surface, c. By directly binding the sample present in the test sample to the CE, a complex is formed containing the sample bound to the biaffinity probe, d. Further flowing the sample, which has been composited with the DAP, in a lateral direction on the surface, thereby causing it to composite with the surface bonding portion (SBM), e. The method for determining the presence and / or amount of the sample in the test sample based on the presence of the biaffinity probe complexed with the SBM.

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