Methods for digital immunosensing of single molecules using a label immobilization and amplification strategy

Abstract

The present invention relates to diagnostic tests and diagnostic techniques. In particular, the present invention relates to a method for determining the presence of an analyte in a sample, comprising: (a) coupling the sample to (i) a first binding agent capable of specifically binding the analyte and immobilized on a solid support; and (ii) a second binding agent capable of specifically binding the analyte and reversibly binding at least one detectable label, wherein the first binding agent and / or the second binding agent are linked to a linking agent capable of covalently linking the reversibly bound at least one detectable label to the solid support when the first binding agent and the second binding agent are in physical proximity. (b) contacting a solid support with a first binding agent, the test substance, and a second binding agent for a time and under conditions that allow specific binding of the test substance to the first binding agent and the second binding agent, such that a complex of the first binding agent, the test substance, and the second binding agent is formed, and that allow covalent attachment of at least one detectable label to the solid support; (b) removing the complex of the first binding agent, the test substance, and the second binding agent from the solid support, such that a solid support remains having at least one detectable label covalently attached thereto; and (c) detecting the covalently attached at least one detectable label on the solid support, thereby determining the test substance. The present invention also relates to a device for determining an analyte suspected to be present in a sample, to the use of the device of the present invention for determining an analyte suspected to be present in a sample in said sample, and to a kit for determining an analyte suspected to be present in a sample.

Description

[Technical field]

[0001] The present invention relates to diagnostic tests and techniques. In particular, the present invention relates to a method for determining the presence of an analyte in a sample, comprising: (a) coupling the sample to a first binding agent capable of specifically binding to the analyte and immobilized on a solid support; and (ii) a second binding agent capable of specifically binding to the analyte and reversibly binding to at least one detectable label, wherein the first binding agent and / or the second binding agent are linked to a linking agent capable of covalently linking the reversibly bound at least one detectable label to a solid support when the first binding agent and the second binding agent are in physical proximity. The present invention relates to a method for determining an analyte suspected to be present in a sample, comprising: (a) contacting a solid support with a first binding agent and a second binding agent for a time and under conditions that allow specific binding of the analyte to the first binding agent and the second binding agent, such that a complex of the first binding agent and the second binding agent is formed, and that allows at least one detectable label to be covalently attached to the solid support; (b) removing the complex of the first binding agent, the analyte and the second binding agent from the solid support, such that a solid support remains having at least one detectable label covalently attached thereto; and (c) detecting the at least one covalently attached detectable label on the solid support, thereby determining the analyte. The present invention also relates to a device for determining an analyte suspected to be present in a sample, a use of the device of the present invention for determining an analyte suspected to be present in a sample in said sample, and a kit for determining an analyte suspected to be present in a sample. [Background technology]

[0002] Immunoassays are widely used for various diagnostic purposes. Several setups for immunoassays have been developed. One of the most common immunoassays is the enzyme-linked adsorbent immunoassay (ELISA).

[0003] In ELISA, a liquid sample containing or suspected to contain an analyte of interest is applied to a stationary solid phase with special binding properties due to the presence of antibodies or antibody-like molecules such as aptamers. After sample application, several reagents are added sequentially, incubated and washed to perform and stop the anytical detection reaction. After all these steps are performed, a physical or chemical property of the setup, usually in the liquid phase, changes and can be detected. Typically, an optical change occurs in the final liquid phase, such as the development of a color due to the product of an enzymatic reaction. These changes correlate to the presence or abundance of the analyte of interest present in the investigated sample. The typically quantitative readout is usually based on the detection of the intensity of the transmitted light by spectrophotometry, which involves the quantification of the transmission of light of a particular wavelength through the liquid. The sensitivity of the detection depends on the amplification of the signal during the analytical reaction. Since the enzymatic reaction is a very well-known amplification process, the signal is generated by an enzyme linked to a detection reagent at a constant rate to allow accurate quantification.

[0004] For the ELISA setup, the analyte binding agent, e.g., an antibody, is immobilized on a solid support phase, such as a solid support structure. Usually, the antibody is coated and dried on the transparent bottom and sometimes also the side walls of the wells of an analytical multi-well plate or analytical vial. However, nanoparticles or other beads can also be used as the solid phase for ELISA.

[0005] For research and diagnostic applications, ELISA is often used in the so-called sandwich ELISA format. In the sandwich format, an immobilized capture antibody is used to capture, i.e. specifically bind, the analyte present in the sample applied to said immobilized antibody. After the capture antibody specifically binds to the analyte, the sample material is washed away. In a subsequent step, the analyte bound to the immobilized antibody is incubated with a detection antibody that specifically binds to the analyte or to the complex of the analyte and the capture antibody. The detection antibody typically includes a detectable linker or adapter molecule that allows it to attract such a detectable label from the solution.

[0006] However, in light of the fragile immune complexes required for signal generation and the various components used in such ELISAs in sandwich format, there are many possibilities for undesired binding of detection antibodies and thus the generation of false positive signals.Therefore, sandwich ELISAs used in research often require validation due to the risk of false positive results.In addition, due to the various shortcomings of such fragile multi-component assay formats, there are limitations in terms of sensitivity and specificity. Summary of the Invention

[0007] The technical problem underlying the present invention may be seen as the provision of means and methods for addressing the above-mentioned needs. This technical problem is solved by the embodiments characterized in the following claims and in the specification.

[0008] Accordingly, the present invention provides a method for determining the presence of a test substance suspected of being present in a sample, comprising the steps of: (a) subjecting the sample to (i) a first binding agent capable of specifically binding to the test substance and immobilized on a solid support; and (ii) a second binding agent capable of specifically binding to the test substance and reversibly binding to at least one detectable label, a second binding agent, wherein the first binding agent and / or the second binding agent is linked to a linking agent capable of covalently binding at least one reversibly bound detectable label to a solid support when the first binding agent and the second binding agent are in physical proximity; - allowing specific binding of the test substance to the first binding agent and the second binding agent such that a complex of the first binding agent, the test substance and the second binding agent is formed; and - contacting for a time and under conditions that allow for covalent attachment of at least one detectable label to the solid support; (b) removing the complex of the first binding agent, the test agent, and the second binding agent from the solid support so as to leave a solid support having at least one covalently attached detectable label; and (c) detecting at least one detectable label covalently attached to the solid support, thereby determining the test substance; The present invention relates to a method comprising the steps of: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] It should be understood that, in this specification and claims, "a" or "an" can mean one or more, depending on the context in which it is used. Thus, for example, a reference to "an" item can mean that at least one item can be utilized.

[0010] When used hereinafter, the terms "have", "comprise" or "include" are meant to have an open-ended or closed-ended meaning. Thus, these terms with a closed-ended meaning may refer to a situation in which no other features are present in the described embodiment apart from the features introduced by these terms, i.e., these terms have a closed-ended meaning in the sense of "consisting of" or "consisting essentially of". Terms with a closed-ended meaning refer to a situation in which, in addition to the features introduced by these terms, one or more other features are present in the described embodiment.

[0011] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "typically" and "more typically" or similar terms may be used in conjunction with additional or alternative features without limiting the possibilities for substitution.

[0012] Furthermore, as used herein, the term "at least one" will be understood to mean that one or more of the items followed by the term may be used in accordance with the present invention. For example, if the term indicates that at least one item should be used, this may be understood as one item or more than one item, i.e., two, three, four, five, or any other number. Depending on the item to which the term refers, a person skilled in the art will understand what upper limit, if any, the term may refer to.

[0013] The method according to the invention may consist of steps (a) and (c) as described above or may comprise further steps, such as pre-treating or isolating the sample before step (a) and / or after step (c), and may further comprise one or more steps of evaluating the determined test substance, for example by comparing it with a reference, in order to provide a diagnostic, prognostic, environmentally-related, agriculturally-related or analytically-related conclusion depending on the purpose of the determination of the test substance.

[0014] As used herein, the term "determining" encompasses any kind of qualitative or quantitative determination of a test substance. A qualitative determination aims to determine the presence or absence of a test substance in a sample, whereas a quantitative determination aims to determine the amount of the test substance. A quantitative determination, i.e., a determination of the amount, includes determining an absolute amount (e.g., total amount by weight or number of molecules present in a sample) or a relative amount (e.g., amount relative to a sample volume (concentration) or a classification such as a score (e.g., "high", "low", etc.). Typically, determining a test substance includes determining the presence or absence of said test substance or the amount of said test substance. More typically, the amount is quantitatively determined by using "digital" detection as specified elsewhere herein.

[0015] The term "test substance" as referred to herein relates to any type of molecule or agent suitable for determination by the method of the present invention. It will be understood that such molecule or agent may have a size and / or structure that allows the binding of the first and second binding agents referred to herein. Furthermore, there may be an upper size limit since the first and second binding agents and linking agents must be able to perform their functions as described in detail elsewhere herein. Typically, the test substance referred to herein is a small molecule such as a protein, peptide, nucleic acid, e.g. DNA or RNA, or lipid or metabolite, e.g. polyketides, including flavonoids and isoflavonoids, isoprenoids, including terpenes, sterols, steroids, carotenoids, xanthophylls, carbohydrates, phenylpropanoids, alkaloids, benzenoids, indoles, porphyrins, hormones, vitamins, cofactors, lignins, glucosinolates, purines, pyrimidines, nucleosides, nucleotides, alcohols, alkanes, alkenes, alkynes, aromatic compounds, ketones, aldehydes, carboxylic acids, esters, amines, imines, amides, cyanides, amino acids, thiols, thioesters, phosphate esters, sulfate esters, thioethers, sulfoxides or ethers. Small molecules can be, for example, toxins. However, the method of the present invention can also be used to determine viruses or even bacterial cells as test substances. Test substances determined by the present invention can also be molecules present in environmental samples and can be useful, for example, as indicators of environmental pollution, agriculture or other environmental conditions. Typically, the test agent is a protein, peptide, virus, bacterial cell or a small molecule, preferably a small molecule toxin.

[0016] As used herein, the term "sample" relates to any portion or aliquot of a composition that contains or is suspected to contain a test substance to be determined. Such a sample may typically be a biological sample isolated from an organism, such as a body fluid or a biopsy sample, or a composition that contains an organism, such as cultured cells. Typically, said biological sample is investigated by the method of the invention for medical purposes, such as the diagnosis or prediction of a disease or condition. The sample may also be an environmental sample or an artificial sample. An environmental sample may originate from any non-biological natural source, e.g. a solution present in the environment, such as water, or a composition, such as soil. An artificial sample may be a sample obtained from an artificial source, e.g. a product composition that may be manufactured or an intermediate composition that may occur during the manufacturing process of a product. Such an artificial sample may be investigated, for example, for quality control purposes or to determine the amount of a particular component. Typically, said sample according to the method of the invention is a biological sample, preferably a body fluid or a biopsy sample.

[0017] The term "first binding agent" refers to a molecule that can specifically bind to the test substance, i.e., does not bind to and therefore does not cross-react with other molecules other than the test substance suspected to be present in the sample. Specific binding can in principle be tested by techniques well known in the art, including screening assays to identify agents that specifically bind to the test substance from a library containing different candidate agents.

[0018] The first binding agent according to the present invention is immobilized on a solid support. The immobilization of the first binding agent can be permanent or reversible. Permanent immobilization can be achieved by techniques well known in the art. However, it is envisaged that the immobilized first detection agent can be released from the solid support. In the case of antibodies or other proteins or peptides used as the first detection agent, this can be achieved by specific proteolytic cleavage. In the case of nucleic acid-based aptamers, removing the first binding agent immobilized on the solid support from the solid support can be achieved by nuclease digestion. Typically, the immobilization is a reversible immobilization, for example, immobilization achieved by nucleic acid hybridization, such as L-DNA capture.

[0019] Preferably, the molecule that can be used as the first binding agent capable of specifically binding to the desired test substance can be an antibody. The antibody as a binding agent as meant in accordance with the present invention includes all types of antibodies that preferably specifically bind to the test substance. Preferably, the antibody of the present invention can be a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody, or any fragment of such an antibody that can still specifically bind to the test substance. Such fragments included in the term antibody as used herein include bispecific antibodies, synthetic antibodies, Fab, F(ab) and the like. 2The antibody or fragment thereof that specifically binds to the desired test substance can generally be obtained by using the methods described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by techniques including fusion of mouse myeloma cells to spleen cells derived from an immunized mammal, preferably an immunized mouse (Koehler 1975, Nature 256, 495, and Galfre 1981, Meth. Enzymol. 73, 3). The skilled artisan is well aware of how specific binding can be tested by techniques well known in the art, such as immunoassays, cell sorting, or immunological or biochemical techniques including Western blotting or plasmon resonance measurements.

[0020] Furthermore, the molecule that can be used as a first binding agent, preferably capable of specifically binding to a desired analyte, can be an aptamer. Aptamers as binding agents according to the present invention can be oligonucleotide acid or peptide molecules that bind to a specific target analyte (Ellington 1990, Nature 346(6287):818-22). Bock 1992, Nature 355(6360):564-6). Oligonucleotide acid aptamers are engineered by repeated selection or the so-called systematic evolution of ligands by exponential enrichment (SELEX technique). Peptide aptamers usually contain a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint increases the binding affinity of peptide aptamers into the nanomolar range. The variable peptide loop length is preferably composed of 10-20 amino acids, and the scaffold can be any protein with improved solubility and compactness, for example thioredoxin-A. The selection of peptide aptamers can be carried out using different systems, including for example the yeast two-hybrid system (see for example Hoppe-Seyler 2000, J Mol Med. 78(8):426-30). Any fragment of said aptamer that is still capable of specifically binding to the test substance is also encompassed according to the present invention. Said fragments can be used in isolated form or can be part of a fusion molecule, i.e. a molecule that comprises said aptamer fragment as well as other moieties such as linker moieties or adapter molecules. The skilled person is well aware how specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurements.

[0021] Preferably, the molecule that can be used as the first binding agent capable of specifically binding to the desired test substance can be a receptor molecule. The receptor molecule as a binding agent referred to according to the present invention is typically a protein that specifically binds to a ligand and is activated upon ligand binding to exert its biological function. Such receptor molecules or fragments thereof that can still specifically bind to a ligand can also be used as a binding agent according to the present invention for the ligand as a test substance, or for molecules derived from the ligand but still capable of being bound by the receptor molecule or fragments thereof, such as antagonistic or agonistic mutants of the ligand. Preferably, the receptor molecule envisaged as a binding agent according to the present invention can be a transmembrane receptor protein (such as a G protein-coupled receptor, such as a metabolic receptor, an enzyme-linked receptor, such as a receptor tyrosine kinase, such as a growth factor receptor, an immune receptor, such as a viral receptor, a cell surface antigen, or a T cell receptor, such as CD4, CD3, or CD8), an MHC protein, a cell adhesion molecule (such as an integrin, cadherin, selectin, or syndecan), a neuronal receptor, or a pathogen receptor (such as a Toll-like receptor). However, the receptor molecule may also be a nuclear receptor protein, such as a nuclear hormone receptor, for example a glucocorticoid receptor, a retinoic acid receptor or a thyroid hormone receptor. The skilled artisan is well aware of receptor molecules or fragments thereof that specifically bind to the test substance or fragments thereof to be measured. Furthermore, specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurements.

[0022] More preferably, the molecule that can be used as the first binding agent that can specifically bind to the desired test substance can be a ligand molecule. The ligand molecule as a binding agent referred to according to the present invention is typically a protein or peptide that specifically binds to a receptor molecule and is activated when the receptor binds to said receptor molecule. Such a ligand molecule or a fragment thereof that can still specifically bind to a receptor molecule can also be used as a binding agent according to the present invention for the receptor as a test substance, or a molecule derived from the receptor molecule but still able to be bound by the ligand molecule or a fragment thereof, such as a soluble mutant of the receptor. Furthermore, the ligand can also be any antigen that can be used to determine a specific antibody in a biological sample. Preferably, the ligand molecules envisaged as binding agents according to the present invention may be peptide hormones, neurotransmitters, growth factors (such as angiopoietins, BMPs, neutrophil factors, EGF, epiphrin, EPO, FGF, GDNF, GDF, insulin or insulin-like growth factors), TGF, neutrophils, VEGF, cytokines (such as interleukins, interferons, lymphokines, monokines, colony-stimulating factors or chemokines), extracellular matrix proteins (such as fibronectin, vitronectin, collagen, ankyrin or laminin), etc. The skilled person is well aware of ligand molecules or fragments thereof that specifically bind to the test substance or fragments thereof to be measured. Furthermore, specific binding can be tested by techniques well known in the art, such as plasmon surface resonance measurements.

[0023] More preferably, the first binding agent can be a designed ankyrin repeat protein (DARPin). DARPins are engineered antibody-mimicking proteins that can be designed to achieve highly specific and high affinity target protein binding. They are derived from natural ankyrin repeat proteins. Typically, DARPins contain at least three repeat modules, of which the most N-terminal and the most C-terminal modules (also called "caps") protect the hydrophobic core of the protein (Binz 2003, Journal of Molecular Biology.332(2):489-503).

[0024] Typically, the first binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof. More typically, the second binding agent is an antibody or fragment thereof or an aptamer.

[0025] As used herein, the term "solid support" refers to a solid composition of matter that can serve as a basis for immobilizing molecules, particularly the first binding agent and the detectable label. The solid support may comprise inorganic or organic compounds or both. Typically, inorganic compounds suitable for solid supports may be selected from the group consisting of silica, porous glass, aluminosilicates, borosilicates, metal oxides (e.g., aluminum oxide, iron oxide, nickel oxide) and clays containing one or more of these. The solid support may also comprise a conductive compound such as a metal or graphite. Preferably, such a solid support may be an electrode, a semiconductor, a plasmon resonator, or any type of electrical circuitry. Alternatively, the solid support may comprise an organic compound such as a cross-linked polymer. Non-limiting examples of suitable cross-linked polymers may be selected from the group consisting of polyamides, polyethers, polystyrenes and mixtures thereof. The skilled artisan is well aware how to select a suitable solid support based on the type of sample to be investigated, the method envisaged for the detection of the analyte, and the type of the first molecule used to detect the analyte.

[0026] Typically, the solid support comprises a linker on its surface to which a label can be covalently attached by a linking agent. Typically, the analog used on the surface of the solid support can be an immobilized peptide that can function as a K tag for transglutaminase.

[0027] However, the solid support envisaged according to the present invention is typically an electrode or a planar waveguide. Also, typically, the solid support is preferably a bead, well or a predetermined subdivided detection area of ​​a plasmon resonator. More typically, each bead, well or predetermined subdivided detection area has a predetermined amount of the first binding agent immobilized thereon, preferably up to 3, up to 2 or most preferably 1.

[0028] When determining the test substance, it includes determining the amount of said test substance, typically preferably using a bead, well or a predetermined subdivided detection area of ​​a plasmon resonator, more preferably immobilizing a predetermined amount of a first binding agent. It will be understood that in light of a predetermined ratio of the first binding agent to the bead, well or predetermined subdivided detection area, the signal generated by one or more detectable labels on the bead or in the well or in the predetermined subdivided detection area correlates with the number of initially formed complexes of the first binding agent, the test substance and the second binding agent. Therefore, preferably, the amount of the test substance is determined by counting the beads, wells or predetermined subdivided detection areas that contain at least one covalently attached detectable label. The number of initially formed complexes, and thus the number of test substances, can then be calculated based on the existing relationship.

[0029] The term "second binding agent" capable of specifically binding to a test substance refers to a molecule capable of specifically binding to the test substance when bound to the first binding agent, or to the first binding agent when bound to the test substance, i.e., a molecule that does not bind to and therefore does not cross-react with other molecules other than the test substance suspected to be present in the sample or the complex of the test substance and the first binding agent. Typically, said second binding agent is selected from the group consisting of an antibody or a fragment thereof, an aptamer, a receptor molecule or a fragment thereof, and a ligand molecule or a fragment thereof. More typically, the second binding agent is an antibody or a fragment thereof or an aptamer. Typically, the second binding agent is from the same aforementioned molecular class as the first binding agent. The definitions of the antibody or a fragment thereof, the aptamer, the receptor molecule or a fragment thereof, and the ligand molecule or a fragment thereof made according to the first binding agent apply mutatis mutandis to the second binding agent.

[0030] As used herein, the term "detectable label" relates to a molecule that exhibits a physical or chemical property that can be detected in the context of the method of the invention. Preferably, said property is an optically detectable property, such as radiation, chemiluminescence, fluorescence or FRET emission, an electromagnetic property, such as interference with an electric or magnetic field, detectable radioactivity, or a chemical property, such as the ability to perform or catalyze a specific chemical reaction. The detectable label reversibly binds to the second binding agent. To this end, the detectable label may be a molecule that can reversibly bind to the second binding agent or may be a molecule or moiety that is already part of said second binding agent. Exemplary detectable labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels or fluorescent labels. Enzymatically active labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or luciferase. Suitable substrates for the detection of such enzymatically detectable labels include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). The appropriate enzyme-substrate combination may produce a colored reaction product, fluorescence or chemiluminescence, which can be measured by methods known in the art (e.g., using a light-sensitive film or a suitable camera system). Exemplary fluorescent labels include fluorescent proteins such as GFP, RFP, YFP, BFP or variants thereof, Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes such as Alexa 568. The use of quantum dots as fluorescent labels is also contemplated. Exemplary radioactive labels include 35S, 125I, 32P, 33P, etc. Radioactive labels can be detected by any method known and appropriate, such as a light-sensitive film or a phosphor imager. Exemplary magnetic labels include iron-platinum nanoparticles, iron nanoparticles, nickel nanoparticles, or cobalt nanoparticles.Typically, the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a radioactive label, a magnetic label, and an electrochemical label. Typically, the detectable label comprises a linker that can be covalently linked to a solid support by a linking agent.

[0031] The term "linking agent" referred to herein relates to an agent capable of covalently binding a detectable label to a solid support. It will be understood that the choice of linking agent used in the method of the present invention depends on the chemical nature of the detectable label and the solid support. The linking agent may be linked to the first binding agent and / or the second binding agent directly or via a linker molecule. The linking agent may be covalently or reversibly linked. Typically, the linking agent can be attached by the linker only after the complex of the first binding agent, the test substance and the second binding agent is formed. In such a case, a split aptamer may be used in which a portion of the aptamer binds to the first binding agent and a second portion of the aptamer binds to the second binding agent. Each aptamer portion alone cannot bind to the linking agent. However, once the complex of the first binding agent, the test substance and the second binding agent is formed, the aptamer specifically binds to the linking agent. Such split aptamers are known in the art (Walter 2017, Nano Lett 17, 2467-2472). Alternatively, when a cross-linking enzyme is used as a linking agent, the first and second binding agents may be linked to different parts of the enzyme that require each other to be present in order to be functionally active. Typically, the linking agent may also be a covalent cross-linking enzyme, preferably a transglutaminase or a sortase, or the linking agent comprises a covalent cross-linking chemical moiety. The skilled person is well aware how to select a linking agent taking into account the chemical nature of the detectable label and / or the solid support.

[0032] Preferably, transglutaminase can be used as a linking agent. Transglutaminase is a cross-linking enzyme that catalyzes the formation of an isopeptide bond between a γ-carboxamide group in the side chain of a glutamine residue and an ε-amino group in the side chain of a lysine residue or a primary amine residue in a peptide or protein, thereby releasing ammonia (EC number EC2.3.2.13). Typically, the transglutaminase envisaged according to the present invention can transfer a Q-Tag on a detectable label to a K-tag peptide immobilized as a linker on a solid support. Transglutaminase is found in various organisms, including bacteria, mammals and humans. In humans, the following transglutaminases have been reported, among others: factor XIII, keratinocyte transglutaminase, tissue-type transglutaminase, epidermal transglutaminase, prostate transglutaminase, TGM X, TGM Y, TGM Z, protein 4.2.

[0033] Preferably, sortases may also be used as linking agents. Sortases are bacterial enzymes that act as transpeptidases (EC number 3.4.22.70). In particular, they cleave substrate proteins or peptides at a conserved cleavage site and catalyze the formation of an amide bond between the carboxy terminus resulting from the cleavage and a free amino group of another protein or peptide, typically a peptidoglycan. Preferably, the transpeptidase activity of sortases can be used such that the cleavage site is added to the C-terminus of a first protein and the oligoglycine motif is added to the N-terminus of a second protein to which it is ligated. Upon addition of sortase to the protein mixture, the two peptides are covalently linked via a native peptide bond. The term includes full-length sortase proteins as well as fragments thereof that have sortase activity. Typically, sortases suitable according to the invention include sortase A, sortase B, sortase C, and D-type sortases.

[0034] Furthermore, the linking agent may preferably comprise a covalent cross-linking chemical moiety. Typically, a chemical moiety suitable as a linking agent is present on the first binding agent and / or the second binding agent, which together chemically catalyze or allow the covalent attachment of the detectable label to the solid support. For example, the detectable label may be non-covalently attached to the complex, for example by an antibody or aptamer acting as a linking agent, in that it is functionally close to the solid support upon formation of the complex of the first binding agent, the test substance, and the second binding agent. The chemical moiety present on the solid support and the chemical moiety present on the detectable label then interact with each other such that a covalent bond is formed between the detectable label and the solid support. Alternatively, the first chemical moiety present on the second binding agent may be functionally close to the solid support presenting a second chemical moiety that is modified upon complex formation to become a third chemical moiety. The third chemical moiety may then be covalently attached to the chemical moiety of the detectable label. Chemical moieties suitable for such an approach may be those applicable to click chemistry reactions such as cycloadditions, e.g., Huisgen 1,3-dipolar cycloadditions, thiol-ene reactions, Diels-Alder reactions, inverse electron demand Diels-Alder reactions, nucleophilic substitutions, carbonyl chemistry-like formation of ureas, or addition reactions to carbon-carbon double bonds such as dihydroxylations or thiol-yne reactions to alkynes. Typically, such reactions are carried out as biorthogonal reactions, i.e., the reactants of the reaction must be kinetically, thermodynamically, and metabolically stable prior to reaction in the system and must not interfere with other reactions or components of the system. Both reactants must interact specifically with each other, i.e., no cross-reactivity should occur in the system. The reaction conditions of the reactants must not interfere with the stability of other components or reactions in the system.

[0035] In step (a) of the method of the invention, a sample containing or suspected of containing the analyte to be determined is contacted with an arrangement comprising a first binding agent capable of specifically binding the analyte, immobilized on a solid support, said arrangement may be provided as a sensor according to the invention, as described elsewhere herein.

[0036] As used herein, the term "contacting" refers to bringing the aforementioned components into physical proximity so that the first and / or second binding agents, if present in the sample, can bind to the test substance. It will be understood that the binding of the first binding agent to the test substance and the binding of the second binding agent to the test substance or to a complex of the first binding agent and the test substance, may require time and application of appropriate conditions. The skilled person is well aware of what time is required and what conditions need to be applied to achieve binding. For example, the sample and the binding agent may be dissolved or mixed with a buffer that adjusts the salt concentration and / or the pH value. It will be understood that suitable buffers and other auxiliary components that may be applied depend on the binding agent used and the chemical nature of the test substance to be determined.

[0037] In step (a) of the method of the present invention, the first binding agent and the test substance are also contacted with a second binding agent that can specifically bind to the test substance and can reversibly bind to at least one detectable label. The second binding agent can be applied after the sample is contacted with the first binding agent immobilized on the solid support. Alternatively, the sample can be contacted with the first binding agent together with the second binding agent, i.e., both components can be applied simultaneously to the solid support and the immobilized first binding agent. As a further alternative, the second binding agent and the sample can be mixed, and the resulting mixture is then applied to the solid support containing the immobilized first binding agent.

[0038] Furthermore, in step (a) of the method of the invention, a detectable label is applied to the sample comprising the first binding agent and the second binding agent, which can be done before or after the components are contacted with each other, as described, or together when the sample is contacted with the first binding agent and / or the second binding agent.

[0039] When the first and second binding agents are in physical proximity, the first and / or second binding agents applied in the method of the present invention are linked to a linking agent that can covalently bind at least one reversibly linked detectable label to a solid support.As mentioned above, this is the case when the linking activity of the linking agent is present so that a complex between the first binding agent, the test substance and the second binding agent is formed and the detectable label can be covalently bound to the solid support.Therefore, step (a) of the method further requires that it is carried out under both a time and conditions that allow the specific binding of the test substance to the first and second binding agents, so that a complex of the first binding agent, the test substance and the second binding agent is formed, and so that at least one detectable label is covalently bound to the solid support.

[0040] The detectable label can be covalently attached to a solid support or a similar molecule immobilized on a solid support. Depending on the chemical nature of the detectable label, the skilled artisan will be well aware of suitable linkers for the detectable label or means for directly linking the detectable label to a solid support.

[0041] As a result of the above-mentioned activity occurring during step (a) of the method of the present invention, at least one detectable label is covalently bound to the solid support.It will be understood that when enzyme is used as a linking agent, as a result of one complex formation caused by the presence of one test substance, two or more detectable labels are immobilized on the solid support by covalent binding.Therefore, the event of complex formation caused by the presence of one test substance molecule in sample can be amplified to cause various immobilization of detectable label molecules on the solid support, which allows easy and accurate detection.

[0042] In step (b) of the method of the invention, the complex of the first binding agent, the test substance and the second binding agent is removed from the solid support leaving a solid support having at least one detectable label covalently attached thereto.

[0043] As used herein, the term "removing" refers to any means of removing the entire complex formed in step a or a part thereof from the solid support such that the linking agent is no longer able to immobilize it by reversible binding to the detectable label and / or covalent binding to the solid support. Typically, the removal referred to herein is achieved by washing the solid support with a washing solution such as a buffer applying conditions that dissolve the complex or its components. Alternatively or additionally, temperature or other physical forces, such as shaking or centrifugation, may be applied. However, it will be understood that the covalently attached detectable label remains attached on the solid support and is not affected by the measurement used to remove the complex. The appropriate means for removing the complex typically depends on the nature of the immobilization of the first binding agent to the solid support. If the first binding agent is immobilized, for example, by nucleic acid hybridization, applying temperature and / or salt that promotes dissociation of the hybridized nucleic acid strands may be used to remove the complex. When the first binding agent is an immobilized antibody or fragment thereof, a receptor protein or fragment thereof, or a ligand protein or fragment thereof, proteolytic digestion using a protease can be used to cleave the immobilized antibody or fragment thereof, the receptor protein or fragment thereof, or the ligand protein or fragment thereof from the solid support.Similarly, when a nucleic acid-based aptamer is used as the first binding agent, nuclease digestion can be used to cleave the aptamer from the solid support.

[0044] As a result of step (b), the solid support is free of intact complexes, and only the covalently attached detectable label remains, which represents a previous complex formation event. Furthermore, the detectable label that may generate non-specific noise signals, as well as secondary binders and other components that may interfere with proper signal detection induced by the covalently attached detectable label, are also largely removed.

[0045] In step (c) of the method of the present invention, at least one covalently bound detectable label on the solid support is detected, thereby determining the analyte.

[0046] As used herein, the term "detecting" relates to identifying the presence, absence and / or amount of a labeled molecule covalently bound to a solid support by measuring a physical, chemical, physicochemical and / or biological property of said labeled molecule. It will be understood that a detectable label typically generates a signal that can be measured. The signal intensity and / or duration typically indicates the amount of detectable labeled molecule present on the solid support. Depending on the chemical nature of the detectable label, the signal may be an active signal, such as the emission of light or other radiation, or a passive signal, i.e. a signal induced as a response to an external stimulus, such as applying an electromagnetic field, radiation, etc. The skilled person is well aware how the measurement of such a signal induced by a detectable labeled molecule can be performed, and it will be understood that the detection method used according to the method of the present invention depends on the detectable label used. Preferred measurement methods according to the present invention also include chemiluminescence measurements, fluorescence measurements, FRET measurements, electrochemiluminescence measurements, mass measurements including mass spectrometry, magnetic or electric field based measurements using e.g. ChemFET or other electrode arrangements, radioimmunoassay measurements, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) measurements, scintillation proximity assay measurements, quantum dot technology measurements, turbidimetric measurements or nephelometric measurements.

[0047] The detectable label may also be detected indirectly, i.e. by using a further labeling molecule capable of specifically binding to the immobilized detectable label on the solid support. Such a further labeling molecule may be applied to the solid support after step (b) has been performed and before step (c). It will be understood that upon specific binding of one or more of said further labeling molecules to the detectable label(s) on the solid support, said further labeling molecules may be detected in step (c) by measuring a physical, chemical, physicochemical and / or biological property of said labeling molecule. Furthermore, further molecules capable of specifically binding to the immobilized detectable label may also serve as anchors for other labeling molecules. Suitable molecules that may function as further labeling molecules or anchor molecules may also be antibodies, aptamers or other molecules that allow specific binding of a target as described elsewhere herein. Suitable labels for such further molecules are those described elsewhere herein for detectable labels.

[0048] In the method of the present invention, the first binding agent and the second binding agent can be used to determine different test substances. In such a case, it will be understood that when complexes for such different test substances are formed, each complex must have a different linking agent to provide different linking activity for different detectable labels. For example, test substance A is determined by using a first binding agent linked to linking agent A and a second detectable label A, and then when complex A is formed, detectable label A is covalently attached to a solid support, and test substance B is subsequently or simultaneously determined by using a first binding agent linked to linking agent B and a second detectable label B, and then when complex B is formed, detectable label B is covalently attached to a solid support. Typically, the detectable labels A and B present on the solid support when test substances A and B are present in the sample being investigated can be detected by using detectable physical, chemical, physicochemical and / or biological properties that are different between the detectable labels.

[0049] In a particular embodiment of the method of the present invention, the solid support is preferably a bead, well or a predetermined subdivided detection area of ​​a plasmon resonator. Preferably, each bead, well or predetermined subdivided detection area has a predetermined amount of a first binding agent immobilized thereon. The method is particularly useful for determining an analyte and comprises determining the amount of the analyte. Typically, the amount is determined by counting beads, wells or predetermined subdivided detection areas that contain at least one covalently attached detectable label. According to this particular embodiment, the presence or absence or amount of detectable label covalently attached to the bead, well or predetermined subdivided detection area is detected for each bead, well or predetermined subdivided area. The number of positive beads, wells or predetermined subdivided areas is then determined. A positive bead, well or predetermined subdivided area is one that exhibits a detectable label or exhibits a detectable label that induces a measurable signal of a predetermined intensity or duration, for example a measurable signal above a predetermined intensity threshold or above a predetermined time window. Based on the positive beads, wells or predefined subdivided regions and the predefined amount of the first binder immobilized on each of the beads, wells or predefined subdivided regions, the amount of the test substance that caused the covalent binding of the detectable label to the bead, well or predefined subdivided region can be determined, for example, by calculation. For example, if each bead, well or predefined subdivided region ideally contains one first binder, the presence of one molecule of the test substance in the sample will generate one complex that causes the covalent binding of at least one detectable label to the well, bead or predefined subdivided region. Thus, when the presence of detectable label(s) on the bead, well or predefined subdivided region is determined, this presence reflects the presence of one test substance present in the sample. Thus, the use of a predefined amount of the first binder on an isolated article such as a bead, well or any other predefined subdivided region allows for the quantitative or semi-quantitative determination of the test substance molecules present in the sample. This technique is also called "digital" detection.

[0050] Advantageously, according to the present invention, it has been found that the use of covalent attachment of detectable labels to solid supports as a result of immune complex formation in a sandwich assay format can be used to improve sensitivity and suppress undesired background noise. According to the present invention, the covalent attachment of detectable labels to solid supports occurs only when an immune complex is formed comprising a first binding agent, e.g. an antibody, an analyte to be determined, a second binding agent, e.g. an antibody, and the first binding agent and the second binding agent due to their participation in the complex acquire or activate a linking agent that allows the covalent attachment of detectable labels to the solid support. Thereby, a single immune complex formation event due to the presence of a single analyte molecule in the sample to be investigated causes the covalent attachment of several detectable labels to the solid support, thus enhancing the signal induced by the detectable labels that represents the presence of said analyte molecule. The components that cause noise can be removed after the detectable label(s) are efficiently covalently attached to the solid support. Depending on the nature of the solid support, it is possible to essentially isolate the complex formation event. For example, when beads are used as solid supports with a single first binding agent immobilized on their surface that initiates complex formation in the presence of one analyte molecule, if the beads after complex formation and removal of noise components contain one or more detectable labels covalently bound to their surface, the labeled beads represent one analyte molecule present in the sample. In such a "digital" detection format, the number of labeled beads can be used to count the analyte molecules present in the sample.

[0051] Thanks to the present invention, it is possible to determine the analyte in a sample with ultimate sensitivity at the molecular number level. Due to the covalent binding of the detectable label to the solid support, noise-causing components such as unbound detectable label and / or first and second binding agents can be efficiently removed by vigorous washing or other treatments.

[0052] The present invention also provides an apparatus for determining an analyte suspected to be present in a sample, comprising: (a) a sensor element, (i) a first binding agent capable of specifically binding to the test substance, the first binding agent being immobilized on a solid support; and (ii) a second binding agent capable of specifically binding to the test substance and reversibly binding to at least one detectable label, a second binding agent, wherein the first binding agent and / or the second binding agent is linked to a linking agent capable of covalently binding at least one reversibly bound detectable label to a solid support when the first binding agent and the second binding agent are in physical proximity; A sensor element including: (b) a detector for detecting at least one detectable label covalently attached to the solid support; and The present invention relates to an apparatus comprising:

[0053] As used herein, the term "device" refers to a system comprising the above-mentioned components operatively linked to each other to allow the determination of an analyte by the method of the present invention.

[0054] The sensor element typically comprises a reaction zone having said first binding agent capable of specifically binding to the analyte and immobilized on a solid support that is in contact with a sample containing the analyte or suspected of containing the analyte. In addition, in the reaction zone, a second binding agent, a detectable label and conditions can be applied that (i) allow specific binding of the analyte to the first binding agent and the second binding agent such that a complex of the first binding agent, the analyte and the second binding agent is formed, and (ii) allow covalent binding of at least one detectable label to the solid support.

[0055] Additionally, the reaction zone allows for the application of conditions to remove the complex of the first binding agent, the test agent, and the second binding agent from the solid support, such that a solid support remains having at least one covalently attached detectable label. As described elsewhere herein, such conditions may include washing with a washing solution, such as a suitable buffer or other solvent, and / or application of temperature or other physical forces.

[0056] The reaction zone may directly allow sample application or may be connected to a loading zone where the sample is applied. In the latter case, the sample may be actively or passively transported to the reaction zone via a connection between the loading zone and the reaction zone. Furthermore, the reaction zone is also connected to a detector. The connection is such that the detector can detect the detectable label covalently attached to the solid support. The appropriate connection depends on the technique used to measure the presence or amount of the detectable label(s). For example, for optical detection, light transmission may be required between the detector and the reaction zone, and for electrochemical determination, a fluid connection may be required, for example, between the reaction zone and an electrode.

[0057] The detector is adapted to detect the presence or amount of the covalently bound detectable label on the solid support. Depending on the detectable label, different detection techniques can be applied.

[0058] Optically detectable labels may be determined by measuring luminescence, fluorescence, FRET, polarization, refraction, etc. Exemplary optical detectors may be photomultipliers, phototubes, ionization detectors, active pixel sensors, phototransistors, photodiodes, quantum dot photoconductors or photodiodes, photovoltaic cells, semiconductor detectors, thermal detectors, photochemical detectors, and the like.

[0059] Electrochemically detectable labels can be determined by electrode configurations such as thin-layer electrodes suitable for voltammetric and typically amperometric measurements. The detector typically comprises at least two electrodes, at least one of which is the so-called working electrode. The electrodes can be made of all conventional electrode materials such as metals, noble metals, alloys or graphite, preferably noble metals such as gold or palladium or graphite. The various electrodes of the sensor can be made of the same or different materials. More typically, the electrodes are made of palladium.

[0060] It will be appreciated that the detected presence, absence or amount of detectable label covalently attached to the solid support may then be transmitted to an evaluation unit, which may preferably include a data processing element such as a computer having an implemented algorithm for determining the presence or amount of an analyte in the sample based on the presence, absence or amount of the detected detectable label(s).

[0061] Such an implemented algorithm can evaluate the measurement signal induced from the detectable label(s) for signal intensity, signal duration and other predefined parameters. Based on said evaluation, truly positive signals can be identified and verified, and noise signals can be identified and ignored for further evaluation. The skilled person is well aware of which algorithms can be used and how they can be implemented in the device of the present invention.

[0062] Furthermore, the evaluation unit may also include an implemented algorithm for identifying individual beads, wells, or predefined subdivided detection areas, counting beads, wells, or predefined subdivided detection areas that contain at least one covalently attached detectable label, and thereby determining the amount of the test substance.

[0063] The present invention generally relates to the use of the device of the present invention for determining an analyte suspected to be present in said sample. More typically, the device is used to determine the analyte by carrying out the method of the present invention as described in more detail elsewhere herein.

[0064] The present invention further provides a kit for determining the presence of a test substance suspected of being present in a sample, comprising: (a) a first binding agent capable of specifically binding to a test substance and immobilized on a solid support; (b) a second binding agent capable of specifically binding to the test substance and reversibly binding at least one detectable label, wherein the first binding agent and / or the second binding agent is linked to a linking agent capable of covalently binding the at least one reversibly bound detectable label to a solid support when the first binding agent and the second binding agent are in physical proximity; (c) a detectable label, and (d) an agent for removing the complex of the first binding agent, the test agent, and the second binding agent from the solid support, so as to leave a solid support having at least one detectable label covalently attached thereto. The present invention relates to a kit comprising:

[0065] As used herein, the term "kit" refers to a collection of the above-mentioned components, typically provided separately or in a single container. The container also typically contains instructions for carrying out the method of the invention. These instructions may be in the form of a manual or may be provided by a computer program code that, when executed on a computer or data processing device, is capable of carrying out or supporting the determination of fibrinogen referred to in the method of the invention. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., compact disc) or directly on the computer or data processing device, or may be provided in a download format, such as a link to an accessible server or cloud. Furthermore, the kit may typically include a test substance solution with a standardized amount or other reference amount for calibration or validation. The kit according to the invention may also include further components necessary for carrying out the method of the invention, such as washing solutions, solvents and / or reagents necessary for the detection of the detectable label. Furthermore, it may partially or entirely include the device of the invention.

[0066] The following embodiments are specific preferred embodiments contemplated in accordance with the present invention, all definitions apply mutatis mutandis to the explanation of terms above.

[0067] Embodiment 1. A method for determining an analyte suspected to be present in a sample, comprising: (a) subjecting the sample to (i) a first binding agent capable of specifically binding to the test substance, the first binding agent being immobilized on a solid support; and (ii) a second binding agent capable of specifically binding to the test substance and reversibly binding to at least one detectable label, a second binding agent, wherein the first binding agent and / or the second binding agent is linked to a linking agent capable of covalently binding the at least one reversibly bound detectable label to the solid support when the first binding agent and the second binding agent are in physical proximity; - allowing specific binding of the test substance to the first binding agent and the second binding agent such that a complex of the first binding agent, the test substance and the second binding agent is formed; and - contacting for a time and under conditions that allow for covalent binding of said at least one detectable label to said solid support; (b) removing the complex of the first binding agent, the test agent, and the second binding agent from the solid support so as to leave the solid support having the at least one detectable label covalently attached thereto; (c) detecting the at least one detectable label covalently attached to the solid support, thereby determining the analyte; A method comprising:

[0068] Embodiment 2. The method of embodiment 1, wherein the test agent is a protein, peptide, virus, bacterial cell, or small molecule, preferably a small molecule toxin.

[0069] Embodiment 3. The method of embodiment 1 or 2, wherein the first binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

[0070] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the second binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

[0071] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the linking agent is a covalent cross-linking enzyme, preferably a transglutaminase or a sortase.

[0072] Embodiment 6. The method of any one of embodiments 1 to 4, wherein the linking agent comprises a covalent cross-linking chemical moiety.

[0073] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a radioactive label, a magnetic label, and an electrochemical label.

[0074] Embodiment 8. The method of embodiment 7, wherein the detectable label comprises a linker that can be covalently linked to the solid support by the linking agent.

[0075] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the solid support comprises a linker on its surface to which the label can be covalently attached by the linking agent.

[0076] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the solid support is an electrode or a planar waveguide.

[0077] Embodiment 11. The method according to any one of embodiments 1 to 9, wherein the solid support is preferably a bead, a well or a predetermined subdivided detection area of ​​a plasmon resonator.

[0078] Embodiment 12. The method of embodiment 11, wherein each bead, well, or predefined subdivided detection area has a predefined amount of a first binding agent immobilized thereon.

[0079] Embodiment 13 The method of embodiment 12, wherein determining the test substance comprises determining the amount of the test substance.

[0080] Embodiment 14. The method of embodiment 13, wherein the amount is determined by counting beads, wells, or predefined subdivided detection areas that contain at least one covalently attached detectable label.

[0081] Embodiment 15. The method of any one of embodiments 1 or 12, wherein determining the test substance comprises determining the presence or absence of the test substance.

[0082] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the sample is a biological sample, preferably a body fluid or a biopsy sample.

[0083] Embodiment 17. An apparatus for determining an analyte suspected to be present in a sample, comprising: (a) a sensor element, (i) a first binding agent capable of specifically binding to the test substance, the first binding agent being immobilized on a solid support; and (ii) a second binding agent capable of specifically binding to the test substance and reversibly binding to at least one detectable label, a second binding agent, wherein the first binding agent and / or the second binding agent is linked to a linking agent capable of covalently binding the at least one reversibly bound detectable label to the solid support when the first binding agent and the second binding agent are in physical proximity. A sensor element including: (b) a detector for detecting the at least one detectable label covalently attached to the solid support; and An apparatus comprising:

[0084] Embodiment 18. Use of the device of embodiment 17 for determining, in said sample, an analyte suspected to be present in the sample.

[0085] Embodiment 19. A kit for determining a test substance suspected to be present in a sample, comprising: (a) a first binding agent capable of specifically binding to a test substance and immobilized on a solid support; (b) a second binding agent capable of specifically binding to the test substance and reversibly binding at least one detectable label, wherein the first binding agent and / or the second binding agent is linked to a linking agent capable of covalently linking the at least one reversibly bound detectable label to the solid support when the first binding agent and the second binding agent are in physical proximity; (c) a detectable label, and (d) an agent for removing the complex of the first binding agent, the test agent, and the second binding agent from the solid support, so as to leave a solid support having at least one detectable label covalently attached thereto. The present invention relates to a kit comprising:

[0086] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification. [Brief description of the drawings]

[0087] [Figure 1]1 shows a schematic configuration of a sandwich assay for carrying out the method of the present invention. On the solid support, there is a linker for immobilizing a first binding agent, for example, a first antibody that can specifically bind to an analyte of interest. This linker, for example, a nucleic acid (LLNA), can be bound to a moiety or adapter on the first binding agent to immobilize the first binding agent on the solid support. The first binding agent, for example, a first antibody, specifically binds to the analyte of interest. The analyte of interest when bound by the first binding agent is specifically bound by a second binding agent, for example, a second antibody. The second binding agent is linked to a linking agent, for example, transglutaminase. Once part of the immune complex, the second binding agent brings the linking agent into proximity with the solid support. The linking agent, for example, transglutaminase, covalently binds a detectable label to the surface of the solid support. This can be achieved by using a Q tag on the label molecule that is transferred and covalently linked to a K tag present as a linker on the solid support. A detectable label that is covalently attached to a solid support is subsequently detected in the methods of the invention after the immune complexes have been removed, for example by a washing step. [Figure 2A] Schematic of the measured signal from the covalently bound detectable label after removal of immune complexes. Only the signal from the detectable label covalently bound in close proximity to the former immune complex is detected as a true signal with a certain intensity. The noise signal induced by potentially non-specific binding or associated detectable label molecules remaining after washing is below the threshold. [Figure 2B] (A) Schematic of the underlying molecular mechanism on the solid support corresponding to the measured signal. [Figure 3A] The undesired second binding agent adjacent to the solid support may also be covalently bound to some detectable label. [Figure 3B] The covalently bound detectable label may remain after extensive washing, but is significantly less than the label that is covalently bound to the solid support as a result of proper immune complex formation. Unwanted covalently bound detectable label results in a noise signal. [Figure 4]A plasmonic surface with predefined subdivided areas for detecting single immune complex formation using CCD camera readout.

Claims

1. 1. A method for determining a test substance suspected of being present in a sample, comprising: (a) subjecting the sample to (i) a first binding agent capable of specifically binding to the test substance and immobilized on a solid support; and (ii) a second binding agent capable of specifically binding to the test substance and reversibly binding to at least one detectable label, a second binding agent linked to a linking agent capable of covalently binding the at least one reversibly bound detectable label to the solid support when the first binding agent and the second binding agent are in physical proximity; - allowing specific binding of the test substance to the first binding agent and the second binding agent such that a complex of the first binding agent, the test substance and the second binding agent is formed; and - contacting for a time and under conditions that allow covalent binding of said at least one detectable label to said solid support; (b) removing the complex of the first binding agent, the test agent, and the second binding agent from the solid support such that the solid support remains having the at least one detectable label covalently attached thereto; (c) detecting the at least one detectable label covalently attached to the solid support, thereby determining the analyte; A method comprising:

2. The method of claim 1 , wherein the first binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

3. The method of claim 1 or 2, wherein the second binding agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a receptor molecule or fragment thereof, and a ligand molecule or fragment thereof.

4. The method of claim 1 or 2, wherein the linking agent is a covalent cross-linking enzyme, preferably a transglutaminase or a sortase.

5. The method of claim 1 or 2, wherein the linking agent comprises a covalent cross-linking chemical moiety.

6. 3. The method of claim 1 or 2, wherein the detectable label is selected from the group consisting of a fluorescent label, a chemiluminescent label, a radioactive label, a magnetic label, and an electrochemical label.

7. The method of claim 6 , wherein the detectable label comprises a linker that can be covalently linked to the solid support by the linking agent.

8. The method of claim 1 or 2, wherein the solid support comprises a linker on its surface to which the label can be covalently attached by the linking agent.

9. The method according to claim 1 or 2, wherein the solid support is preferably a plasmon resonator bead, a well or a defined subdivided detection area.

10. 10. The method of claim 9, wherein each bead, well, or predetermined subdivided detection area has a predetermined amount of the first binding agent immobilized thereon.

11. The method of claim 10 , wherein determining the test substance comprises determining an amount of the test substance.

12. The method of claim 11, wherein the amount is determined by counting beads, wells, or pre-defined subdivided detection areas that contain at least one covalently attached detectable label.

13. 1. An apparatus for determining an analyte suspected to be present in a sample, comprising: (a) a sensor element, (i) a first binding agent capable of specifically binding to the test substance and immobilized on a solid support; and (ii) a second binding agent capable of specifically binding to the test substance and reversibly binding to at least one detectable label, a second binding agent linked to a linking agent capable of covalently binding the at least one reversibly bound detectable label to the solid support when the first binding agent and the second binding agent are in physical proximity; A sensor element including: (b) a detector for detecting the at least one detectable label covalently attached to the solid support; and An apparatus comprising:

14. 14. Use of the device according to claim 13 for determining an analyte suspected to be present in said sample.

15. 1. A kit for determining a test substance suspected of being present in a sample, comprising: (a) a first binding agent capable of specifically binding to the test substance, the first binding agent being immobilized on a solid support; (b) a second binding agent capable of specifically binding to the test substance and reversibly binding at least one detectable label, wherein the second binding agent is linked to a linking agent capable of covalently binding the at least one reversibly bound detectable label to the solid support when the first binding agent and the second binding agent are in physical proximity; (c) a detectable label, and (d) an agent for removing the complex of the first binding agent, the test agent, and the second binding agent from the solid support, such that the solid support remains having the at least one detectable label covalently attached thereto. Including the kit.

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