An immunoassay reagent, kit and method for improving the anti-hooking ability of an assay reagent

Abstract

This application relates to an immunoassay reagent, a kit, and a method for enhancing the anti-hooking ability of the reagent. The reagent includes a biomolecule, a marker, and a Catcher-Tag system. In the Catcher-Tag system, a Tag binds to the biomolecule, and a Catcher binds to the marker. The specific binding between the Catcher and the Tag in the Catcher-Tag system allows the biomolecule to bind directionally to the marker. This technical solution can increase the content of specific antigens / antibodies in the reagent and enhance their activity, thereby comprehensively improving the anti-hooking ability of the reagent and increasing the accuracy of the test results.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to an immunoassay reagent, a kit, and a method for enhancing the anti-HOOK ability of the reagent. Background Technology

[0002] Immunoassay is a method based on the high affinity reaction between antigens and antibodies to quantitatively or qualitatively determine the concentration or relative concentration of an analyte (such as an antigen or antibody) in a sample. It is mainly used in disease diagnosis, health monitoring, prognosis assessment, and drug development.

[0003] The main analytical methods for immunoassay include sandwich assays, indirect assays, and capture assays. Sandwich assays include double-antibody sandwich assays and double-antigen sandwich assays. The principle of sandwich assays is to utilize two different specific antigens / antibodies to bind to two different epitopes of the antibody / antigen to be tested in the sample, forming a "sandwich" structure, thereby detecting the concentration of the antibody / antigen to be tested in the sample. This method has high sensitivity and specificity, and the reaction principle is simple and easy to operate.

[0004] In immunoassay systems, when the concentration of the antigen / antibody is within a certain range, the reaction curve between its concentration and the detection signal value is linear. However, when a certain concentration threshold is exceeded, the detection signal value decreases with increasing concentration, causing the entire reaction curve to exhibit a hook shape, known as the HOOK effect. The presence of the HOOK effect can lead to false negatives and affect the accuracy of the test results. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides an immunoassay reagent, a kit, and a method for enhancing the anti-HOOK capability of the reagent. This method can increase the content of specific antigens / antibodies in the reagent and enhance their activity, thereby comprehensively improving the anti-HOOK capability of the reagent and increasing the accuracy of the test results.

[0006] The first aspect of this application provides an immunoassay reagent, including a biomolecule, a marker, and a Catcher-Tag system; in the Catcher-Tag system, a Tag binds to the biomolecule, and a Catcher binds to the marker, thereby enabling the biomolecule to bind directionally to the marker through the specific binding between the Catcher and the Tag.

[0007] In some embodiments, the Catcher-Tag system is selected from the Spy Catcher-Tag system containing signal molecules and the Catcher-Tag system.

[0008] In some preferred embodiments, the signaling molecule is linked to the Catcher protein of the Catcher-Tag system; more preferably, to the N-terminus and / or C-terminus of the Catcher protein.

[0009] In some preferred embodiments, the signaling molecule is co-expressed with the Catcher protein.

[0010] In some embodiments, the signaling molecules introduced at the N-terminus and C-terminus of the Catcher protein may be the same or different.

[0011] In some embodiments, the signaling molecule is selected from basic amino acid molecules or tag proteins containing basic amino acid residues.

[0012] In some embodiments, the basic amino acid is selected from lysine or arginine.

[0013] In some embodiments, the basic amino acid is selected from monomeric molecules, dimer molecules, or polymeric molecules containing one type of basic amino acid sequence, or from mixed polymeric molecules containing at least two types of basic amino acid sequences.

[0014] In some embodiments, the molecular weight of the tag protein is 0.5 kDa to 50 kDa.

[0015] In some embodiments, the tag protein contains 1 to 50 basic amino acid residues.

[0016] In some embodiments, the proportion of basic amino acid residues in the tag protein is 0.05 to 5 wt%.

[0017] In some embodiments, the isoelectric point of the tag protein is between 4 and 7.5.

[0018] In some embodiments, the tag protein is selected from at least one of Flag-tag, Avi-tag, Myc-tag, Strep-tagII, and Arg-tag.

[0019] In some embodiments, the biomolecule is co-expressed with the tag.

[0020] In some implementations, the marker is attached to the N-terminus and / or C-terminus of the Catcher.

[0021] In some embodiments, the marker binds to a lysine / arginine residue at the end of the catcher.

[0022] In some embodiments, the marker is a luminescent microsphere filled with a luminescent composition, and the biomolecule is an antigen / antibody capable of reacting with the target molecule to be tested.

[0023] In some preferred embodiments, the luminescent microspheres are modified with active groups, which bind to the Catcher; more preferably, the active groups are at least one of carboxyl, amino, aldehyde, and thiol groups.

[0024] In some embodiments, the marker is biotin, and the biomolecule is an antigen / antibody capable of generating an immune response with the target molecule to be tested.

[0025] A second aspect of this application provides an immunoassay kit, comprising: The receptor reagent comprises luminescent microspheres and a first biomolecule; Biotin reagent, which contains a second biomolecule and biotin; The first biomolecule and the second biomolecule can specifically bind to different epitopes of the target molecule, respectively; the first biomolecule and the luminescent microspheres bind directionally through the Catcher-Tag system, and / or the second biomolecule and biotin bind directionally through the Catcher-Tag system.

[0026] In some embodiments, the Catcher-Tag system in the receptor reagent or biotin reagent contains lysine or arginine residues.

[0027] The third aspect of this application provides a method for enhancing the anti-HOOK ability of homogeneous immunoluminescent assay reagents, which uses a Catcher-Tag system to directionally bind biomolecules and markers.

[0028] The fourth aspect of this application provides the application of the above-mentioned reagents or kits in homogeneous immunoluminescence detection.

[0029] Specifically, the detection method includes: adding the above-mentioned receptor reagent and biotin reagent to the sample to be tested, so that the biomolecules therein can react with the target molecule in the sample to be tested; adding the donor reagent (containing photosensitive microspheres and avidin bound to them) to obtain an immune complex; detecting the detection signal generated by the immune complex; and determining whether the sample to be tested contains the target molecule and / or the content of the target molecule in the sample to be tested based on the intensity of the detection signal.

[0030] The technical solution provided in this application can include the following beneficial effects: By introducing a novel antigen-antibody conjugation technology into the immunoassay reagent, namely, achieving the targeted binding of biomolecules such as specific antigens / antibodies to markers through the Catcher-Tag system, the effective region—that is, the region that can specifically bind to the target antibody / antigen—is fully displayed, thereby improving the utilization rate of specific antigens / antibodies in the reagent, enhancing the reagent's anti-hooking ability, reducing the probability of false negatives, and improving the accuracy of the test results. It is suitable for accurate detection of both high-concentration and low-concentration samples.

[0031] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0032] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0033] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0034] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0035] I. Terminology The term "biomolecules" in this article refers broadly to all types of molecules unique to living organisms, all of which are organic compounds. Based on molecular weight, they are divided into two main categories: biomacromolecules and biosmall molecules. Biomacromolecules generally have a molecular weight of over 10,000 and include proteins, nucleic acids, and polysaccharides. Biosmall molecules generally have a molecular weight below 1,000. The basic building blocks of biomacromolecules with biological activity, such as amino acids, small peptides, oligopeptides, oligosaccharides, and oligonucleotides, are biosmall molecules. Vitamins (such as biotin and biotin derivatives), minerals, plant secondary metabolites and their degradation products (such as aglycones, flavonoids, glycosides, and alkaloids) are also biosmall molecules.

[0036] The term "label" as used herein refers to substances used in immunoassays to label biomolecules (e.g., antibodies), enabling them to be recognized by specific detection methods. These labels typically bind to biomolecules via chemical bonding. The labels can be substances capable of generating detectable signals, such as light or radioactive signals, under certain conditions; they can also be substances capable of amplifying the detectable signal-biomolecule complex by aggregating the signal; or they can be carriers containing substances capable of generating or amplifying detectable signals. Specifically, the labels may include, but are not limited to, radioactive isotopes such as the radionuclide iodine-125 (¹²). 5 I) Enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP), fluoresceins such as fluorescein isothiocyanate (FITC) and tetraethylrhodamine (RB200), chemiluminescent compositions such as luminol and its derivatives and acridinium esters, colloidal gold, and signal amplification systems such as biotin in the biotin-avidin system. The labeled substances may also include inorganic / organic luminescent microspheres prepared by physical adsorption, embedding or grafting, copolymerization, or other methods.

[0037] The terms “combination,” “connection,” and “coupling” as used in this article refer to the union between two substances caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, or interactions including but not limited to salt bridges and water bridges.

[0038] The specific binding described in this article refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, is the conformational correspondence between the reactants in the response.

[0039] The directional binding described in this article refers to the phenomenon where two or more molecules bind to each other in a specific direction and at specific sites. This binding is not random, but rather based on the molecular structure, chemical properties, and the specificity of the interaction. For example, the antigen-binding site on an antibody molecule can recognize and bind to specific epitopes on an antigen molecule; this binding exhibits high specificity and directionality.

[0040] The Catcher-Tag system described in this article is a protein covalent linking technology, comprising two parts: a Tag and a Catcher, each consisting of multiple amino acid residues. Covalent linking of proteins is achieved through a specific reaction between the Tag and the Catcher. The Tag-Catcher system may include, but is not limited to, the Spy Catcher-Tag system and the Snoop Catcher-Tag system.

[0041] The Spy Catcher-Tag system described in this paper is developed based on the CnaB2 domain of the fibronectin FbaB from Streptococcus pyogenes. The Spy Tag is a short peptide containing 13 amino acid residues, and the Spy Catcher is a protein containing 138 amino acid residues. During the linkage process, the aspartic acid in the Spy Tag and the lysine in the Spy Catcher spontaneously react to form a heteropeptide covalent bond, catalyzed by the glutamate adjacent to the lysine. The Catcher protein in the Tag-Catcher system can exist as a monomer or a multimeric protein. A multimeric protein is a protein composed of two or more polypeptide chains, which can be identical or different, linked together by covalent or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.).

[0042] The Snoop Catcher-Tag system described in this article was developed from the fimbriae protein RrgA of Streptococcus pneumoniae. The D4 domain of RrgA generates a stable isopeptide bond through an E803 catalytic reaction between a lysine (K742) and an asparagine (N854) that are spatially adjacent.

[0043] The signaling molecules described in this article refer to a class of molecules that can be fused with target molecules (such as proteins, cells, etc.) for expression. These signaling molecules include, but are not limited to, amino acids, peptides, and proteins. These signaling molecules can be used to construct fusion proteins through in vitro DNA recombination technology with the target protein's gene.

[0044] Co-expression, as described in this article, refers to fusing proteins from two or more genes that require co-expression, enabling simultaneous expression. The co-expression linkage can be achieved by linking the coding sequences of two or more genes together using gene recombination technology to form a fusion gene. This fusion gene, upon expression, produces a fusion protein containing different functional domains encoded by multiple genes. Alternatively, the co-expression linkage can be achieved using technologies such as pClick (see "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021 Aug 27; 11(18): 9107-9117. doi: 10.7150 / thno.62444.) to link two or more peptides, proteins, etc., with different or identical gene sequences, forming a fusion protein. Multiple genes in the fusion protein can be expressed simultaneously.

[0045] The basic amino acids mentioned in this article refer to amino acids whose number of amino groups that can be hydrolyzed exceeds the number of carboxyl groups that can be hydrolyzed, resulting in an alkaline solution. Examples of such basic amino acids include lysine (Lys), arginine (Arg), and histidine (His).

[0046] The monomer molecules described herein refer to small molecule compounds containing a single set of sequences. Specifically, the monomer molecules applicable to this invention can be amino acid molecules composed of a single set of amino acid residues.

[0047] The dimer molecules described herein are compounds composed of two sets of identical or different sequences. Dimer molecules applicable to this invention can be compounds composed of two sets of identical amino acid residues.

[0048] The multimer molecules described herein refer to compounds composed of multiple groups of identical or different sequences. Multimer molecules applicable to this invention can be compounds composed of two or more different groups of amino acid residues, or compounds composed of two or more identical groups of amino acid residues.

[0049] The S Catcher described herein is a Spy Catcher, Snoop Catcher, or other Cater protein that can form isopeptide bonds with the corresponding tag without a signaling molecule. The Cater Pro described herein is a S Catcher protein that contains a signaling molecule, such as a tag protein containing a linker peptide (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5), which is linked to the C-terminus or N-terminus of the S Catcher via the linker peptide.

[0050] The tag protein described in this article refers to a polypeptide or protein molecule that is fused with a target protein (such as a Catcher in a Cater-Tag system) using in vitro DNA recombination technology for expression, detection, purification, labeling, and ligation of the target protein. Tag proteins can be selected from commonly used protein tags, such as 6xHIS, Flag, Strep, Arg, Avi, VSV-G, GST, MBP, NusA, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, SUMO, etc.; or they can be constructed through gene editing to create polypeptide sequences containing specific amino acids.

[0051] The S Tag in the S Tag fusion antigen / antibody described in this article can be a Spy Tag, Snoop Tag, or other Tag protein that can form an isopeptide bond with the corresponding Caterer.

[0052] The luminescent microparticles described herein refer to polymeric microparticles filled with a luminescent composition, capable of reacting with reactive oxygen species to generate detectable light signals. Luminescent microspheres may also be called acceptor microspheres or luminescent microspheres. In some specific embodiments of the invention, the luminescent composition undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acrylamide, aryl vinyl ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. In other specific embodiments of the invention, the luminescent composition may further include europium complexes; more preferably, the europium complex is MTTA-EU. 3+ The luminescent microparticles can introduce active groups onto their surface through chemiluminescence. These active groups include, for example, carboxyl (-COOH), amino (-NH2), thiol (-SH), and aldehyde (-CHO). The active groups can be introduced onto the surface of the luminescent microparticles through chemical grafting, where a compound containing the active group reacts with the microparticles to attach the active groups to the surface of the luminescent microparticles.

[0053] The photosensitive microparticles described herein refer to polymeric microparticles filled with photosensitizers that can generate reactive oxygen species upon photoexcitation. These can also be called donor microspheres or photosensitive microspheres. Solutions containing such photosensitive microparticles can be called photosensitive solutions or universal solutions. The photosensitizers can be those known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, but are not limited to these. The photosensitive microparticles can also be filled with other sensitizers; non-limiting examples include certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species.

[0054] The term "reactive oxygen species" as used in this article refers to a general term for oxygen-containing and reactive substances in the body or natural environment. It primarily refers to excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH), as well as nitric oxide and reactive oxygen species). 1 O2), etc.

[0055] The microparticles described herein can be of any size and shape, expandable or non-expandable, porous or non-porous, and have any density, but preferably close to that of water. They are preferably buoyant in water and are composed of transparent, partially transparent, or opaque materials. The microparticles can be solids (such as polymers, metals, glass, organic or inorganic substances such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and siliceous fluids), vesicles (such as synthetic phospholipids, or natural substances such as cells and organelles). A non-limiting example of microparticles suitable for use in this invention is carboxylated polystyrene latex microspheres.

[0056] The target molecules described in this article refer to all kinds of molecules present in biological fluids, including both macromolecules and small molecules. Target molecules include, but are not limited to, proteins, hormones, antibodies, or antigens. Examples of biological fluids include blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion.

[0057] The term "test sample" as used in this article refers to a mixture that may contain the target molecule. Before use, the test sample can be diluted with a diluent or buffer solution to obtain a solution that may contain the target molecule. For example, to avoid the hook effect, the target molecule can be diluted with a sample diluent before detection on the instrument. In this case, any diluted solution that may contain the target molecule is referred to as the test sample.

[0058] The term "antibody" as used herein is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin.

[0059] The antigens described herein refer to substances capable of inducing antibody production, and can be classified into complete antigens and incomplete antigens (haptens). These antigens can be natural antigens extracted from pathogens or animal tissues, or recombinant antigens with specific antigenic properties prepared through genetic engineering techniques. Where necessary, antigens can be further conjugated to other components, such as specific binding pairing members, for example, biotin or avidin.

[0060] The epitopes described in this article, also known as antigenic determinants, are specific chemical groups or regions on the surface of antigen molecules that can be specifically recognized and bound by receptors or antibodies of immune cells. They are a crucial part of the immune response triggered by antigens, determining the specificity of the interaction between antigens and immune cells or antibodies.

[0061] The biotin described in this article is widely found in animal and plant tissues. Its molecule has two ring structures: an imidazoline ring and a thiophene ring, with the imidazoline ring being the primary site for binding to avidin. The biotin can be activated by active groups such as amino, carboxyl, thiol, and NHS-active esters. Activated biotin can then couple with almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents.

[0062] The avidin described herein is a protein secreted by Streptomyces. The streptavidin molecule consists of four identical peptide chains, each capable of binding one biotin, with a molecular weight of 65 kDa. Each antigen or antibody can simultaneously couple multiple biotin molecules, thereby creating a "tentacle effect" with avidin to enhance analytical sensitivity. Where necessary, any reagent used in this invention, including antigens, antibodies, receptors, or donors, can be conjugated to any member of the biotin-streptavidin specific binding pair, as required.

[0063] II. Specific Implementation Plan This application will now be described in more detail.

[0064] In homogeneous immunoluminescence assays, taking photo-induced chemiluminescence as an example, two specific antigens / antibodies are typically used to detect the target antibody / antigen in the sample. These two specific antigens / antibodies are coated on luminescent microspheres and labeled with one of their specific binding partners, such as biotin. When the target antibody / antigen is present in the sample, it binds to both the specific antigen / antibody on the luminescent microspheres and the biotin-labeled antigen / antibody. The biotin binds to the avidin-coated, highly photosensitive microspheres, forming a "sandwich" structure. Upon excitation by light, the excited microspheres undergo reactive oxygen species transfer with the luminescent microspheres, triggering a chemiluminescence reaction that generates a detectable light signal. The intensity of this light signal is directly proportional to the concentration of the target antibody / antigen in the sample.

[0065] The inventors of this application discovered in their research that the activity and affinity of specific antigens / antibodies in the detection reagents can affect their ability to specifically recognize and bind to the antibody / antigen to be tested. This results in a large number of antibody / antigen to be tested that fail to produce an immune response, or a large number of immune products that can produce an immune response but fail to trigger a chemiluminescent reaction, leading to a hook effect and affecting the accuracy of the detection results.

[0066] The immunoassay reagent involved in this application includes biomolecules, markers, and a Cater-Tag system. The Cater binds to the marker, and the Tag binds to the biomolecule. The specific binding between the Cater and Tag in the Cater-Tag system enables the biomolecule to bind directionally to the marker.

[0067] This application utilizes the spontaneously formed isopeptide bonds between the Catcher and Tag in the Catcher-Tag system to enable biomolecules to bind stably and irreversibly to the marker, resulting in a biomolecule-marker. This biomolecule-marker has a strong ability to capture or detect target molecules in the sample, improving the utilization rate of biomolecules, ensuring the immune response between the target antigen / antibody and the specific antigen / antibody during the detection process, enhancing the anti-hooking ability of the detection system, reducing the probability of false negative or weak positive test results, and improving the accuracy of the test results.

[0068] In some embodiments of this application, the Catcher-Tag system may be selected from the Spy Catcher-Tag system, Snoop Catcher-Tag system, or other Catcher-Tag systems that can form isopeptide bonds, with or without a signal molecule; preferably, it contains a signal molecule.

[0069] The signaling molecule can be attached to any position in the Catcher-Tag system; preferably, it is attached to the Catcher protein in the Catcher-Tag system; more preferably, it is attached to the N-terminus and / or C-terminus of the Catcher protein. When signaling molecules are introduced at both the N-terminus and C-terminus of the Catcher protein, the introduced signaling molecules may be the same or different.

[0070] In some embodiments of this application, the signaling molecule can bind to the Catcher protein through co-expression or chemical coupling; co-expression is preferred. For example, the sequence of the signaling molecule can be inserted into the beginning or end of the Catcher protein sequence using genetic engineering techniques to construct a fusion expression vector, and then the Catcher protein fused with the signaling molecule can be obtained through methods such as induced expression and purification, so that it can be expressed simultaneously.

[0071] When the introduced signaling molecule is a tag protein, the tag protein can bind to the catcher via a linker peptide at one end. Preferably, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n; more preferably, n is a natural number from 1 to 5.

[0072] In some embodiments of this application, the signaling molecule may be selected from basic amino acid molecules or tag proteins containing basic amino acid residues.

[0073] In some preferred embodiments of this application, the basic amino acid is selected from lysine or arginine. The basic amino acid molecule can be selected from monomeric molecules, dimer molecules, or polymeric molecules containing one type of basic amino acid sequence, or from mixed polymeric molecules containing at least two types of basic amino acid sequences. Specifically, the signal molecule is a monomeric molecule, dimer molecule, or polymeric molecule containing lysine residues or arginine residues; the signal molecule can also be a mixed polymeric molecule containing lysine residues and arginine residues.

[0074] In some embodiments of this application, the tag protein may be selected from at least one of commonly used protein tags, such as solubilization tags, molecular chaperones, enzyme tags, and purification tags. Specifically, the tag protein may be selected from at least one of Flag, Strep, Arg, Avi, VSV-G, GST, MBP, NusA, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, and SUMO; preferably Flag-tag, Avi-tag, Myc-tag, Strep-tag II, or Arg-tag.

[0075] In other embodiments of this application, the tag protein can also be constructed by gene editing into a polypeptide sequence containing a specific amino acid, such as a polypeptide sequence containing lysine or arginine.

[0076] In some embodiments of this application, the molecular weight of the tag protein can be 0.5 kDa to 50 kDa; preferably 15 kDa to 30 kDa.

[0077] In some embodiments of this application, the number of basic amino acid residues in the tag protein can be 1 to 50.

[0078] To further improve the stability and sensitivity of the detection reagents, the proportion of basic amino acid residues in the tag protein is 0.05-5 wt%; preferably 0.2-3 wt%.

[0079] In some embodiments of this application, the isoelectric point of the tag protein is between 4 and 7.5; preferably between 6.5 and 7.5.

[0080] Signal molecules containing lysine and arginine residues can increase the lysine or arginine content on the Catcher protein. This can improve the efficiency of biomolecule coating or labeling, increase the number of binding sites on the Catcher that can bind to the label, enhance the stability of the biomolecule after coating or labeling, improve the stability of the detection reagent, improve the reagent's resistance to the hook effect, and overall improve the sensitivity of the detection reagent and the accuracy of the detection results.

[0081] To obtain a catcher protein containing a signal molecule at its terminal (N-terminus or C-terminus), the catcher gene can be modified. The following describes the method for modifying a catcher protein containing a signal molecule, using lysine, arginine, or a tag protein containing lysine or arginine as an example. The lysine or arginine sequence can refer to a single group, two groups, or more of these amino acid residue sequences: 1) Add a lysine or arginine sequence directly to the N-terminus of the Catcher protein gene sequence; 2) Add a lysine or arginine sequence directly to the C-terminus of the Catcher protein gene sequence; 3) Add lysine or arginine sequences directly to the N-terminus and C-terminus of the Catcher protein gene sequence; 4) Add a tag protein sequence to the N-terminus of the Catcher protein gene sequence; 5) Add a tag protein sequence to the C-terminus of the Catcher protein gene sequence; 6) Add tag protein sequences to the N-terminus and C-terminus of the Catcher protein gene sequence; 7) Add a tag protein sequence, a lysine sequence, and an arginine sequence to the N-terminus and C-terminus of the Catcher protein gene sequence.

[0082] Catcher-Tag systems containing signaling molecules with lysine or arginine residues can bind to specific functional groups on the label during the binding process of biomolecules and labels. This facilitates stable binding between biomolecules and labels, improves the stability of detection reagents, enhances the consistency of detection results, and has significant anti-hooking ability, which can be maintained for a long time.

[0083] In some embodiments of this application, biomolecules and tags are co-expressed. For example, technologies such as pClick can be used to link biomolecules and tags together to form fusion proteins, enabling their simultaneous expression.

[0084] In some embodiments of this application, the marker binds to the N-terminus and / or C-terminus of the catcher. The marker can be linked to the catcher terminus via physical adsorption or chemical bonding. Further, the marker can be linked to a lysine or arginine residue at the catcher terminus. The lysine or arginine residue at the catcher terminus can be part of its own sequence or introduced into the signaling molecule via recombination technology.

[0085] In some specific embodiments of this application, the marker is a luminescent microsphere filled with a luminescent composition, and the biomolecule is an antigen / antibody capable of reacting with the target molecule to be tested. Preferably, the luminescent microsphere is modified with an active group, which binds to the Catcher protein; the active group can be a carboxyl group, amino group, aldehyde group, thiol group, etc.

[0086] The Catcher protein, through its terminal lysine or arginine residues, can bind to luminescent microspheres modified with active groups such as carboxyl or aldehyde groups, resulting in luminescent microspheres coated with the Catcher. The biomolecule, i.e., the antigen / antibody to be coated, can be linked to the Tag through chemical coupling or recombinant technology, and then the natural binding of the Catcher to the Tag yields luminescent microspheres specifically coated with the antigen / antibody.

[0087] When the label is a luminescent microsphere, the antigen / antibody can be directionally coated onto the luminescent microsphere using the Catcher-Tag system. This increases the amount of specific antigen / antibody attached to the surface of the luminescent microsphere and allows the active end of the specific antigen / antibody to be fully exposed. This increases the effective molecular weight of antigen / antibody in the detection reagent that can react with the target molecule, improves the utilization rate of specific antigen / antibody, and thus enhances the anti-hooking ability of the detection reagent and improves the accuracy of the detection results.

[0088] In some specific embodiments of this application, the marker is biotin, and the biomolecule is an antigen / antibody capable of reacting with the target molecule to be tested.

[0089] When the label is biotin, it is directionally labeled onto specific antigens / antibodies using the Catcher-Tag system. This allows the active end of the specific antigen / antibody to be fully displayed, thus preserving the activity of the biotinylated antigen / antibody in the test reagent. This increases the effective molecular weight of antigen / antibody in the test reagent that can react with the target molecule, improves the utilization rate of specific antigen / antibody, and consequently enhances the anti-hooking ability of the test reagent, thereby improving the accuracy of the test results.

[0090] This application also relates to an immunoassay kit, which includes the aforementioned assay reagents. Specifically, it includes a receptor reagent and a biotin reagent. The receptor reagent comprises luminescent microspheres and a first biomolecule, and the biotin reagent comprises a second biomolecule and biotin. The first and second biomolecules are capable of specifically binding to different epitopes of the target molecule in the sample, respectively. The first biomolecule and the luminescent microspheres bind directionally via a Catcher-Tag system, and / or the second biomolecule and biotin bind directionally via a Catcher-Tag system.

[0091] To enhance the anti-hooking capability of detection reagents, the binding of biomolecules and labels in either or both of the receptor and biotinylate reagents can be achieved using the Catcher-Tag system. Luminescent microspheres can be directionally coated with specific antigens / antibodies using the Catcher-Tag system to obtain directionally coated receptor reagents. Similarly, biotin can be directionally labeled onto specific antigens / antibodies using the Catcher-Tag system to obtain directionally labeled biotinylate reagents.

[0092] The aforementioned targeted-coated receptor reagent can be used in combination with a targeted-labeled biotin reagent to detect the target molecule in the sample; alternatively, the aforementioned targeted-coated receptor reagent can be used in combination with a conventionally labeled biotin reagent; or alternatively, the aforementioned targeted-labeled biotin reagent can be used in combination with a conventionally coated receptor reagent.

[0093] In some embodiments of this application, the Catcher-Tag system in the receptor reagent and / or biotin reagent carries a lysine residue or an arginine residue.

[0094] In some embodiments of this application, when the binding of biomolecules to the label in both the receptor reagent and the biotin reagent is achieved through a Catcher-Tag system, the Catcher-Tag systems used for both can be the same or different; preferably, different Catcher-Tag systems can reduce cross-interference between the two reagents in the detection system, thereby further improving the accuracy of the detection results.

[0095] In addition, the kit includes a photosensitive reagent comprising photosensitive microparticles and avidin coated on the surface of the photosensitive microparticles, which can specifically bind to the biotin marker in the biotin reagent.

[0096] This application also relates to a method for enhancing the anti-hooking ability of homogeneous immunoluminescent assay reagents, which utilizes a Catcher-Tag system to directionally bind biomolecules and markers. This allows the active end of the biomolecules to be fully expressed, improving the utilization rate of the biomolecules, i.e., increasing the amount of effective antibodies / antigens in the reagent that can react with the target molecule, thereby enhancing the anti-hooking ability of the assay reagent.

[0097] The reagents described above can be used in homogeneous immunoluminescence detection. For example, they can be used in photochemiluminescence detection.

[0098] When the above reagents are used in a photo-induced chemiluminescence detection platform, the specific detection methods include: S1. Add luminescent reagent and biotin reagent to the sample to be tested, so that the specific antigen / antibody therein can react with the target molecules in the sample to be tested to obtain the reaction solution; S2. Add a photosensitive reagent to the reaction solution to cause the avidin and biotin in it to bind specifically to shorten the distance between the photosensitive microspheres and the luminescent microspheres, thus obtaining a mixed solution. S3. Excite the mixture with light of a specific wavelength and detect the resulting detection signal. Determine whether the sample contains the target molecule and / or the content of the target molecule in the sample based on the intensity of the detection signal.

[0099] In the homogeneous immunoluminescence assay reagent, biotin or luminescent microspheres are used as markers. The specific antigens / antibodies coated on the luminescent microspheres and the biotin-labeled specific antigens / antibodies are biomolecules that can interact with the target molecules. The binding of the markers and biomolecules is achieved through the Catcher-Tag system, which can increase the amount of effective antibodies / antigens in the assay reagent that can react with the target molecules, thereby enhancing the anti-hooking ability of the assay reagent.

[0100] III. Specific Implementation Examples To make the present invention easier to understand, the present application will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not limited to the scope of application of the present application. Unless otherwise specified, the raw materials or components used in the present application can be obtained commercially or by conventional methods.

[0101] The following uses Spy Catcher-Tag as an example to prepare luminescent microspheres with directed antibody coating (receptor reagent R1) and biotinylated antibody reagents with directed labeling (biotin reagent R2). These reagents are then combined with luminescent microspheres with directly coated antibodies (receptor reagent R1) and biotinylated antibody reagents with directly labeled antibodies (biotin reagent R2) to detect the test samples in a photo-induced chemiluminescence detection platform using the sandwich immunoassay principle. The light signal values ​​are detected and compared to determine the detection performance of directed coating and / or directed labeling antibodies.

[0102] Example 1: Preparation of S Catcher (Pro) and Spy Tag fusion antibodies 1. Preparation and purification of Spy Catcher and Spy Catcher Pro (1) Construction of Catcher plasmid expression vector: The expression vector of Catcher (E. coli vector-pet series expression plasmid) was constructed based on the sequence of Spy Catcher (GenBank accession number: JQ478411.1).

[0103] (2) Catcher expression induced by E. coli: The E. coli expression vector was used to induce the expression of Catcher. The culture conditions were as follows: ① Self-induction medium (10g peptone, 5g yeast extract, 1x NPS, 1mM MgCl2, 1x 5052, pH=7.4), with 100nM ampicillin added; ② Induction conditions: overnight culture at 30℃ and 200rpm in a shaker.

[0104] (3) Catcher purification: ① After overnight reaction, centrifuge at 8000 rpm and collect the bacterial pellet; ② After culture, centrifuge the bacterial solution at 8000 rpm for 2 minutes, discard the supernatant, and retain the bacterial cells; ③ Resuspend the bacterial cells in 100 mL of purification loading buffer A (50 mM PBS, 150 mM NaCl, 20 mM ID, pH=7.4), and after the resuspended bacterial solution is homogenized by high pressure homogenizer, centrifuge at 18000 rpm for 40 minutes to separate the supernatant and inclusion bodies; ④ Collect the supernatant, sonicate for 1 minute (sonication conditions: 2s sonication, 3s interval, 60% power) to break the nucleic acid, and filter with a 0.22 μm needle filter after sonication; ⑤ Obtain Catcher protein using conventional Ni-NTA affinity chromatography gravity column purification method, purification buffer: supernatant (soluble protein) of solution A, loading and equilibration buffer (50 mM PBS, 150 mM NaCl, 25 mM imidazole, pH=7.4). 7.4) + B solution supernatant (soluble protein) elution buffer (50mM PBS, 150mM NaCl, 500mM imidazole, pH 7.4), the target protein is obtained by adjusting the imidazole concentration; ⑥ Elution conditions are: 5%, 15%, 50% and 100% of B solution for fractional elution.

[0105] (4) The protein concentration of the collected portion was determined by the BCA method. Then, the portion containing protein was subjected to reduction and non-reduction electrophoresis. A collection tube that matches the molecular weight of the Catcher protein was selected and dialyzed into the subsequently labeled buffer.

[0106] Prepare S Catcher Pro containing the signal molecules in Table 1 following the steps described above: Table 1

[0107] 2. Preparation and purification of S Tag-antibodies The Spy Tag (sequence AHIVMVDAYKPTK) was linked to the antibody using pClick technology to obtain the Spy Tag-linked antibody. The protein concentration of the collected fraction was determined by the BCA method, and the fraction containing protein was then subjected to electrophoresis. Fractions matching the molecular weight of the Tag-antibody were collected and dialyzed into the buffer required for subsequent coupling reactions to obtain the co-expressed STag-antibody.

[0108] Example 2: Preparation of receptor reagents 1. Preparation of directionally coated luminescent microparticles (1) The luminescent microparticles bind to the Catcher ① Dialyze the purified S Catcher / S Catcher Pro to 0.05M CB, pH 9.6 buffer and determine the protein concentration. ② Dialyze 10 mg of luminescent microparticles to 0.05M CB, pH 9.6 buffer by centrifugation. ③ Add a certain amount of Tween-20 to the luminescent microparticle solution and sonicate to disperse (0.8 mg of Tween-20 per 10 mg of luminescent microparticles). ④ After homogenization by sonication, add 0.05-1 mg of S Catcher or S Catcher Pro protein per 10 mg of microparticles according to the coating ratio, vortex to mix, and react overnight at room temperature. ⑤ Remove the reaction tube, add 10 μL of 8 mg / mL NaBH4, vortex to mix, and react for 2 h. ⑥ After the reaction, centrifuge and wash with luminescent buffer. ⑦ Adjust the volume to 20 mg / mL with buffers of 10 mM PBS, 50 mM Tris-HCl, and 50 mM HEPES (pH 6-8).

[0109] (2) Screening of luminescent microparticles FG microparticles coated with S Catcher and S Catcher Pro were reacted with an antibody against the Catcher-purified tag protein (Biotin Anti-6X His tag® antibody, purchased from Abcam) in a photochemiluminescence platform. The reaction system was: 25 μL FG-Catcher (concentration 50 μg / mL) + 25 μL Biotin Anti-6X His tag® antibody (2 μg / mL) + 25 μL 10 mM PBS. After incubation for 17 min, 175 μL of R3 universal photosensitive solution (containing photosensitive microparticles) was added and incubated for 10 min. The readings were then taken.

[0110] Screening for FG-catchers with strong reaction signals indicates that the luminescent microparticles were successfully coated with SCatcher / S Catcher Pro, which was then used for subsequent coupling reactions.

[0111] (3) Conjugation of luminescent microparticles with S Tag-antibody ① Mix the pre-coated S Catcher or S Catcher Pro FG microparticles with S tag-antibody 1 (hereinafter collectively referred to as Ab1-tag) at a mass ratio of FG:Ab1-tag = 10:(0.25~5), with a microparticle concentration of 10 mg / mL. Incubate at room temperature for 1 h. ② Wash with luminescent buffer and remove any possible free antibody by centrifugation. Repeat the centrifugation and washing process three times. Finally, resuspend the microparticles in luminescent buffer and bring the volume to 10 mg / mL to obtain the luminescent microparticles of the directionally conjugated antibody. The directionally coated microparticles are hereinafter referred to as FG-Ab1-tag.

[0112] 2. Preparation of luminescent microparticles by direct coating The process of directly coating antibody Ab1 is the same as the process of targeted antibody coating using Catcher-Tag. Hereinafter, the directly coated microparticles will be referred to as FG-Ab1.

[0113] 3. Prepare R1 reagent containing 50 μg / mL FG-Ab1-tag or FG-Ab1 by mixing the directionally coated antibody-coated microparticles FG-Ab1-tag and the directly coated antibody-coated microparticles FG-Ab1 with luminescent buffer.

[0114] Example 3: Preparation of Biotin Reagent 1. Biotin-labeled antibody (1) Catcher-labeled biotin: Purified S Catcher and S Catcher Pro were dialyzed into a 0.1M NaHCO3, pH=8.3 buffer solution. NHs-LC-LC-Biotin (purchased from Thermo Fisher Scientific) was added at a Catcher to biotin molar ratio of 1:(10~45) and reacted overnight at 4°C. Free biotin was removed by dialysis, desalting or other similar methods, and the product was finally stored in a 0.01M NaHCO3, pH=8.3 buffer solution, and the protein concentration was determined.

[0115] (2) Catcher-antibody conjugation reaction: The labeled S Catcher or S Catcher Pro was mixed with SpyTag-antibody 2 (hereinafter collectively referred to as Ab2-tag) at a molar ratio of 1:(1~4). After the reaction, a second purification was performed using Protein A chromatography to remove free (i.e., uncovalently bonded to the antibody) Catcher, yielding the directionally conjugated biotinylated antibody (Ab2-tag biotin). The protein concentration of the collected fraction was determined by the BCA method, and the fraction containing protein was then subjected to electrophoresis. Collection tubes matching the molecular weight after conjugation were selected and dialyzed into biotinylate storage buffer. The directionally labeled antibody is referred to as Ab2-tag biotin.

[0116] 2. Biotin-labeled antibodies The process of directly labeling biotin with antibodies is the same as the process of targeted labeling with Catcher-Tag. Hereinafter, the directly labeled antibody will be referred to as Ab2 biotin.

[0117] 3. Prepare R2 reagent by mixing the directionally labeled biotinylated antibody Ab2-tag biotin and the directly labeled biotinylated antibody Ab2biotin with biotin buffer to obtain a solution containing 2 μg / mL Ab2-tag biotin or Ab2 biotin.

[0118] Example 4: Comparison of detection performance of coated end reagents Taking the glial fibrillary acidic protein (GFAP) detection reagent as an example, the effective antibody coating amount of FG-Ab1-tag with directional antibody coating and FG-Ab1 with direct antibody coating is qualitatively compared using the secondary antibody.

[0119] 1. Experimental Procedure (1) The receptor reagent R1 and biotin reagent R2 prepared by the methods described in Examples 2 and 3, together with the photosensitive microparticle (purchased from PerkinElmer) universal solution, i.e., reagent R3, were reacted in a photoluminescence platform. The reaction signals of the luminescent microparticles with the antibody-directly coated and the luminescent microparticles with the antibody-directly coated with the biotinylated antibody were compared. The higher the signal, the better the antibody activity after coating.

[0120] In reagent R1, Ab1 in the directionally coated antibody microparticle FG-Ab1-tag and the directly coated antibody microparticle FG-Ab1 is a mouse IgG antibody; in reagent R2, Ab2 in the biotinylated antibody Ab2 biotin is a mouse IgG secondary antibody, both purchased from Invitrogen.

[0121] (2) Reaction system: 25 μL R1 reagent + 25 μL R2 reagent + 25 μL 10 mM PBS, incubated for 17 min, then 175 μL universal photosensitive solution was added and incubated for 10 min. The photoluminescence instrument reading was then taken. The detection results are shown in the table below.

[0122] 2. Experimental Results Table 2

[0123] Note: In FG-I-Ab1-tag, the Catcher is an S Catcher Pro fused with signal molecule I; in FG-II-Ab1-tag, the Catcher is an S Catcher Pro fused with signal molecule II; in FG-III-Ab1-tag, the Catcher is an S Catcher Pro fused with signal molecule III; and in FG-IV-Ab1-tag, the Catcher is an S Catcher Pro fused with signal molecule IV.

[0124] 3. Experimental Data Analysis Combinations ①-⑤ showed good linear correlation with the calibrators, exceeding 0.99. Different proteins fused to Catcher exhibited varying reactivity.

[0125] In combinations ①-④, the coating end all employed isopeptide-coupled antibody technology, but the proteins fused to the Catcher differed. Results showed that S Catcher Pro fused with tag protein II exhibited better reactivity, with higher signal abundance, correlation, and slope. Therefore, the fused signal molecule should ideally contain lysine residues, have a molecular weight between 15kDa and 30kDa, and a pI (isoelectric point) range of 6.5 to 7.5.

[0126] Compared with ⑤, the discrimination of groups ①-④ using targeted conjugated antibodies was significantly improved, especially in both low and high value samples, indicating improved detection accuracy. This suggests that the targeted coating method preserves the antibody activity to the greatest extent.

[0127] Example 5: Comparison of the stability of coated end reagents Comparison of the stability of FG-Ab1-tag with directed antibody coating and FG-Ab1 with direct antibody coating.

[0128] 1. Experimental Procedure (1) Accelerated test at 37℃ using reagent R1. Reagent R1 was placed in an incubator at 37°C for a total of 7 days. On day 0 (D0, before accelerated experiment), day 1 (D1), day 3 (D3), day 5 (D5), and day 7 (D7), reagent R1 was reacted with reagent R2 (directly labeled antibody) in a photoluminescence platform with a universal photosensitive solution for detection. The stability of the luminescent microparticle reagent with directional antibody coating and the luminescent microparticle reagent with direct antibody coating were compared.

[0129] (2) Reaction system: 25 μL R1 reagent + 25 μL R2 reagent + 25 μL 10 mM PBS, incubated for 17 min, then 175 μL universal photosensitive solution was added and incubated for 10 min. The photoluminescence instrument reading was then taken. The detection results are shown in the table below.

[0130] 2. Experimental Results (1) Stability test results of luminescent microparticle reagents for directional coating of antibodies.

[0131] Table 3

[0132] (2) Stability test results of luminescent microparticle reagents directly coated with antibodies.

[0133] Table 4

[0134] 3. Experimental Data Analysis In the accelerated experiment at 37℃, the correlation of the luminescent microparticles with the directional coated antibody showed almost no significant fluctuation, and the average decrease in signal value was less than 10%; while the antibody directly labeled at the coated end showed large signal fluctuations, with the signal value decreasing by up to 23%.

[0135] The stability of the coated end is significantly improved compared to reagents that are directly coated with antibodies after using directional coupling technology.

[0136] Example 6: Comparison of detection performance of labeled reagents The antibody activities of biotin-directed Ab2-tag biotin and biotin-directed Ab2 biotin were qualitatively analyzed using secondary antibodies.

[0137] 1. Experimental Procedure (1) The luminescent microparticle reagent (R1 reagent) for direct-coated antibody prepared as in Example 2, together with the biotin-directed coated antibody and biotin-direct coated antibody reagent (R2 reagent) prepared in Example 3, and photosensitive microparticles (purchased from PerkinElmer) universal solution, were reacted on a photoluminescence platform, and the intensity of the reaction signal was compared. The higher the signal, the better the activity of the labeled antibody. In the R1 reagent, the Ab1 of the luminescent microparticle FG-Ab1 is a mouse IgG secondary antibody; in the R2 reagent, the Ab2 of the biotinylated antibody Ab2-tag biotin and Ab2 biotin is a mouse IgG antibody. (2) Reaction system: 25 μL R1 reagent + 25 μL R1 reagent + 25 μL 10 mM PBS, incubated for 17 min, then 175 μL R3 reagent (universal photosensitive solution) was added and incubated for 10 min, and the reading was taken. The detection results are shown in the table below.

[0138] 2. Experimental Results Table 5

[0139] 3. Experimental Data Analysis Biotin-labeled reagents prepared by direct labeling and antibody labeling using directional conjugation technology showed significant differences in signal abundance and correlation. Compared with direct labeling, directional labeling significantly improved both the detection signal value and correlation, with the correlation reaching 0.999.

[0140] In addition, the signal differentiation of the directionally coupled label is also higher, indicating that the antibody activity is better after directional labeling compared with direct labeling.

[0141] Example 7: Comparison of the stability properties of labeled reagents 1. Experimental Procedure Both directionally labeled Ab2-tag biotin and directly labeled Ab2 biotin were prepared into R2 reagent (containing 2 μg / mL Bio-mouse IgG antibody) with biotin-buffered saline solution at a concentration of 2 μg / mL. The R2 reagent was incubated at 37°C for a total of 7 days. On days 0 (D0, before accelerated experiments), 1 (D1), 3 (D3), 5 (D5), and 7 (D7), the R2 reagent was reacted with the directly antibody-coated luminescent microparticles (R1 reagent) using a universal photosensitive solution in a photochemiluminescence platform to compare the stability of the biotin-directly labeled antibody reagent and the biotin-directly labeled antibody reagent. The detection results are shown in the table below.

[0142] 2. Experimental Results (1) Stability test results of biotin-directed labeled antibody reagent.

[0143] Table 6

[0144] (2) Stability test results of biotin-labeled antibody reagent.

[0145] Table 7

[0146] 3. Experimental Data Analysis In the accelerated experiment at 37℃, the correlation of the biotin-directed labeling antibody reagent showed almost no significant fluctuation, and the average decrease in signal value remained within 10%, while the decrease in signal value of the biotin-directed labeling antibody reagent reached 21%, which was much higher than that of the directed labeling reagent.

[0147] The reagent stability was good after the labeling end adopted the directional coupling technology, which was significantly improved compared with direct labeling.

[0148] Based on the above experimental results, using isopeptide-coupled antibody technology for targeted antibody labeling or coating in a photo-induced chemiluminescence platform can reduce the impact of labeling and coating methods on antibody activity and achieve high detection accuracy. Fusing a II-tagged protein to the catcher followed by biotin labeling or coating it onto an FG can further enhance the activity of the labeled and coated antibodies.

[0149] Example 8: Preparation of CEA reagent The following uses CEA (carcinoembryonic antigen, purchased from Fitzgerald, USA) photochemiluminescence detection reagent as an example to compare the preparation of detection reagents coated or labeled with anti-CEA (carcinoembryonic antigen antibody) through the methods of Examples 1 to 3.

[0150] CEA detection kit assembly: R1 reagent: luminescent microspheres and anti-CEA bound to the luminescent microspheres; R2 reagent: Biotin-labeled anti-CEA; Pair with R3 reagent: photosensitive reagent (streptavidin-photosensitive microsphere solution).

[0151] The specific differences between CEA reagents are shown in Table 8.

[0152] Table 8

[0153] Example 9: Comparison of the anti-hooking ability of coated end reagents 1. Experimental Procedure (1) CEA antigen (purchased from Fitzgerald, USA) was diluted with physiological saline to multiple concentration gradients between 10,000 and 200,000 ng / mL. The information on the CEA detection reagents used is shown in the table below: Table 9

[0154] The CEA antigen dilutions at the above concentration gradients were tested using experimental groups A, B, and C respectively. The concentration at which the chemiluminescence value decreased with increasing concentration was taken as the lowest concentration of antigen at which the hook effect occurred, and this concentration was taken as the HOOK concentration.

[0155] (2) Detection method: On an automated chemiluminescence detection instrument, add 25 μL of the antigen or sample to be tested, 25 μL of reagent 1, and 25 μL of reagent 2 sequentially to one reaction well, and incubate at 37°C for 15 min; then add 175 μL of the universal solution (photosensitive reagent) for the photo-induced chemiluminescence analysis system to the above reaction well, and incubate at 37°C for 10 min; use the detection instrument to read the values, and obtain the chemiluminescence signal values ​​(RLU). The detection results are recorded in the table below.

[0156] 2. Experimental Results Table 10

[0157] 3. Experimental Data Analysis Spy Catcher (Pro)-Tag-mediated coating of R1 reagent can enhance anti-HOOK ability compared to R1 reagent that directly coats antibodies, especially Spy Catcher Pro-Tag, which has a more significant enhancement effect.

[0158] Example 10: Comparison of the anti-hooking ability of labeled reagents 1. Experimental Procedure CEA antigen was diluted with physiological saline to multiple concentration gradients between 10,000 and 200,000 ng / mL. Information on the CEA detection reagents used is shown in the table below. Table 11

[0159] Following the method of Example 9, the CEA antigen dilutions at the aforementioned concentration gradients were tested using experimental groups A, B, and C. The test results are recorded in the table below.

[0160] 2. Experimental Results Table 12

[0161] 3. Experimental Data Analysis Spy Catcher (Pro)-Tag-mediated labeling of R2 reagents can enhance anti-HOOK ability compared to R2 reagents that directly label biotin, especially Spy Catcher Pro-Tag, which has a more significant enhancement effect.

[0162] Example 11: Comparison of the anti-hooking ability of coated and labeled reagents 1. Experimental Procedure CEA antigen was diluted with physiological saline to multiple concentration gradients between 10,000 and 200,000 ng / mL. Information on the CEA detection reagents used is shown in the table below. Table 13

[0163] Following the method of Example 9, the CEA antigen dilutions at the aforementioned concentration gradients were tested using experimental groups A, B, and C. The test results are recorded in the table below.

[0164] 2. Experimental Results Table 14

[0165] 3. Experimental Data Analysis The use of Spy Catcher (Pro)-Tag-mediated coating of R1 reagent and Spy Catcher (Pro)-Tag-mediated labeling of R2 reagent can further enhance the anti-hooking ability of the reagents, especially the Spy Catcher Pro-Tag, which has a more significant enhancement effect.

[0166] Based on the above experimental results, using isopeptide-coupled antibody technology for targeted labeling or coating of antibodies in a photo-induced chemiluminescence platform can reduce the impact of labeling and coating methods on antibody activity and improve the activity of labeled and coated antibodies. Reagents prepared using targeted coupling technology exhibit good reaction correlation, good anti-hooking ability, and high detection accuracy.

[0167] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An immunoassay reagent, characterized in that, It includes biomolecules, markers, and a Catcher-Tag system; in the Catcher-Tag system, the Tag binds to the biomolecule, and the Catcher binds to the marker, so that the biomolecule and the marker are directionally bound through the specific binding between the Catcher and the Tag.

2. The reagent according to claim 1, characterized in that, The Catcher-Tag system is selected from the Spy Catcher-Tag system and the Snoop Catcher-Tag system, which contain signal molecules; Preferably, the signal molecule is linked to the Catcher protein of the Catcher-Tag system; more preferably, it is linked to the N-terminus and / or C-terminus of the Catcher protein. Preferably, the signaling molecule is co-expressed with the Catcher protein.

3. The reagent according to claim 2, characterized in that, The signaling molecule is selected from basic amino acid molecules or tag proteins containing basic amino acid residues. Preferably, the basic amino acid is selected from lysine or arginine; Preferably, the basic amino acid molecule is selected from monomer molecules, dimer molecules or polymer molecules containing one type of basic amino acid sequence, or is selected from mixed polymer molecules containing at least two types of basic amino acid sequences. Preferably, the molecular weight of the tag protein is 0.5 kDa to 50 kDa; Preferably, the number of basic amino acid residues in the tag protein is 1 to 50; Preferably, the proportion of basic amino acid residues in the tagged protein is 0.05~5wt%; Preferably, the isoelectric point of the tag protein is between 4 and 7.

5.

4. The reagent according to any one of claims 1 to 3, characterized in that, The biomolecule is co-expressed with the Tag; Preferably, the marker is attached to the N-terminus and / or C-terminus of the Catcher; Preferably, the marker binds to a lysine / arginine residue at the end of the catcher.

5. The reagent according to any one of claims 1 to 4, characterized in that, The marker is a luminescent microsphere filled with a luminescent composition, and the biomolecule is an antigen / antibody capable of reacting with the target molecule to be tested. Preferably, the luminescent microspheres are modified with active groups, which bind to the Catcher. Preferably, the active group is at least one selected from carboxyl, amino, aldehyde, and thiol groups.

6. The reagent according to any one of claims 1 to 4, characterized in that, The marker is biotin, and the biomolecule is an antigen / antibody capable of generating an immune response with the target molecule to be tested.

7. An immunoassay kit, characterized in that, include: The receptor reagent comprises luminescent microspheres and a first biomolecule; Biotin reagent, which contains a second biomolecule and biotin; The first biomolecule and the second biomolecule can specifically bind to different epitopes of the target molecule in the sample to be tested, respectively; the first biomolecule and the luminescent microspheres bind directionally through the Catcher-Tag system, and / or the second biomolecule and biotin bind directionally through the Catcher-Tag system.

8. The reagent kit according to claim 7, characterized in that, The Catcher-Tag system in the receptor reagent and biotin reagent contains lysine or arginine residues.

9. A method for enhancing the anti-HOOK ability of homogeneous immunoluminescent assay reagents, characterized in that, The Catcher-Tag system enables targeted binding of biomolecules and markers.

10. The reagent as described in any one of claims 1 to 6, or the kit as described in claim 7 or 8, in homogeneous immunoluminescence detection.

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