Immunoassay kits, their preparation methods and applications

Abstract

This application discloses an immunoassay kit, its preparation method, and its application. The immunoassay kit includes a small peptide-coated solid-phase carrier, a bispecific antibody, and a labeled detection antibody. The bispecific antibody includes an anti-small peptide antibody and a linked capture antibody. The anti-small peptide antibody specifically binds to the small peptide, and both the capture antibody and the labeled detection antibody specifically bind to the target analyte and have different antigen recognition epitopes. The highly specific binding of the small peptide to the anti-small peptide antibody achieves efficient immobilization of the bispecific antibody, significantly improving antibody immobilization density and activity retention compared to traditional detection methods. The targeted binding effect of the bispecific antibody amplifies the detection signal while avoiding competitive interference from endogenous biotin, achieving highly sensitive and interference-resistant immunoassay.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, specifically to immunoassay kits, their preparation methods, and applications. Background Technology

[0002] Immunoassay technology is widely used in clinical diagnosis, biomedical research, and food safety testing, among which sandwich immunoassay is a commonly used detection mode. Currently, the mainstream sandwich immunoassay methods for magnetic bead surface modification fall into two main categories: one is direct coating with specific antibodies, achieving capture through the specific binding of antibodies to the analyte; the other is immobilizing biotinylated antibodies on the streptavidin-modified magnetic bead surface via a biotin-streptavidin bridging method. However, direct antibody coating suffers from uneven antibody immobilization efficiency and susceptibility to activity issues. While the biotin-streptavidin bridging method can improve immobilization efficiency, endogenous biotin in serum samples competes with biotin in the detection system for binding to streptavidin, leading to detection interference. Furthermore, both methods have limited signal amplification capabilities and insufficient sensitivity for detecting low concentrations of analytes, making it difficult to meet the clinical needs for trace biomarker detection. Summary of the Invention

[0003] Therefore, it is necessary to provide immunoassay kits, their preparation methods, and applications.

[0004] The first aspect of this application provides an immunoassay kit comprising a small peptide-coated solid-phase carrier, a bispecific antibody, and a marker-labeled detection antibody, wherein the bispecific antibody comprises an anti-small peptide antibody and a linked capture antibody, the anti-small peptide antibody specifically binding to the small peptide, and both the capture antibody and the marker-labeled detection antibody specifically binding to the target and having different antigen recognition epitopes.

[0005] In some embodiments, the small peptide comprises 5 to 20 amino acid residues; optionally, the amino acid sequence of the small peptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0006] In some embodiments, the solid support includes magnetic beads; optionally, the particle size of the magnetic beads is 1 μm to 5 μm.

[0007] In some embodiments, the marker includes one or more of enzyme markers, electrochemiluminescent markers, and fluorescent markers;

[0008] Optionally, the enzyme marker includes at least one of horseradish peroxidase and alkaline phosphatase;

[0009] Optionally, the electrochemiluminescent label includes ruthenium terpyridine;

[0010] Optionally, the fluorescent marker includes at least one of FITC and Cy5;

[0011] In some embodiments, the immunoassay kit further includes one or more of a detection substrate, buffer solution, blocking solution, positive control and negative control.

[0012] A second aspect of this application provides a method for preparing the immunoassay kit described in the first aspect of this application, comprising the following steps:

[0013] The immunoassay kit is prepared by providing a solid-phase carrier coated with the small peptide, the bispecific antibody, and the marker-labeled detection antibody, respectively.

[0014] In some embodiments, the preparation method of the bispecific antibody includes covalently coupling the anti-small peptide antibody and the capture antibody using a cross-linking agent to prepare the bispecific antibody.

[0015] In some embodiments, the crosslinking agent includes one or more of SMCC and SPDP.

[0016] In some embodiments, the concentration ratio of the anti-small peptide antibody to the capture antibody is (0.8~1.2):(0.8~1.2).

[0017] In some embodiments, the conditions for covalent coupling include a temperature of 20°C to 30°C and a time of 0.5h to 1.5h.

[0018] In some embodiments, the preparation method of the bispecific antibody includes constructing a recombinant expression vector containing a variable region gene of an anti-small peptide antibody and a variable region gene of a capture antibody, transfecting it into host cells for culture, and preparing the bispecific antibody.

[0019] In some embodiments, the host cells include one or more of CHO cells and HEK293 cells.

[0020] In some embodiments, the method for preparing the small peptide-coated solid-phase support includes mixing the small peptide and the solid-phase support and incubating them to prepare the small peptide-coated solid-phase support.

[0021] In some embodiments, the surface of the solid support is modified with active groups, including one or more of carboxyl, amino, and epoxy groups.

[0022] In some embodiments, the mass ratio of the small peptide to the solid support is (1~5):10.

[0023] In some embodiments, the incubation conditions include a temperature of 4°C to 37°C and a time of 8 hours to 16 hours.

[0024] A third aspect of this application provides an immunoassay method comprising detecting a target analyte in a sample to be tested using the immunoassay kit described in the first aspect of this application.

[0025] In some embodiments, the immune detection method includes the following steps:

[0026] A solid-phase support coated with small peptides and a bispecific antibody were mixed and incubated to prepare a solid-phase support-small peptide-bispecific antibody complex.

[0027] The sample to be tested and the solid-phase carrier-small peptide-double antibody complex were mixed and incubated to prepare the solid-phase carrier-small peptide-double antibody-target complex.

[0028] The solid-phase carrier-small peptide-biantibody-target complex and the label-labeled detection antibody are mixed and incubated to prepare a sandwich complex;

[0029] Add the detection substrate to initiate the reaction, and then detect the reaction signal.

[0030] In some embodiments, the concentration ratio of the bispecific antibody to the marker-labeled detection antibody is (1~5):1.

[0031] In some implementations, the sample to be tested includes plasma or serum.

[0032] The fourth aspect of this application provides the use of the immunoassay kit described in the first aspect of this application in the preparation of products for detecting tumor markers or myocardial markers.

[0033] The aforementioned immunoassay kit achieves efficient fixation of bispecific antibodies through the highly specific binding of small peptides and anti-small peptide antibodies. Compared with traditional detection methods, it significantly improves antibody fixation density and activity retention rate. The detection signal is amplified through the targeted binding effect of the bispecific antibodies, while avoiding competitive interference from endogenous biotin, thus achieving highly sensitive and interference-resistant immunoassay. Detailed Implementation

[0034] To facilitate understanding of this application, it may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0036] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0037] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0038] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0039] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0040] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0041] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0042] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0043] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0044] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0045] In this application, the terms "room temperature" or "normal temperature" generally refer to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0046] The traditional sandwich immunoassay technique currently has the following limitations:

[0047] 1. Limited detection sensitivity: When directly coating antibodies, the fixation density and activity stability of antibodies on the surface of magnetic beads are poor, and the signal amplification effect of the biotin-streptavidin bridging method is limited, making it difficult to effectively detect low concentrations of analytes;

[0048] 2. Serum samples have significant interference: The biotin-streptavidin bridging system is susceptible to competitive interference from endogenous biotin in the sample, leading to false negatives or inaccurate quantification.

[0049] 3. Low antibody utilization: Direct coating may mask some antibody active sites, and biotinylation may affect the antibody's specific binding ability, leading to a decrease in antibody utilization and increased detection costs.

[0050] 4. Poor detection stability: Directly coated antibodies are prone to detaching from the surface of magnetic beads, and the binding of biotin-streptavidin is greatly affected by the reaction environment, resulting in poor detection repeatability.

[0051] Therefore, to improve the sensitivity of immunoassay and eliminate interference from endogenous biotin in serum samples, this application provides at least an immunoassay kit, its preparation method, and its application.

[0052] In this application, "bispecific antibody" refers to a bifunctional antibody or a bispecific antibody, which is an antibody that simultaneously possesses the specificity to bind to two antigens and can bind to small peptides and analytes, respectively.

[0053] In this application, "small peptide" refers to a short peptide chain composed of 5-20 amino acid residues, which has the characteristics of high specificity, small molecular weight and easy synthesis.

[0054] In this application, "HRP" refers to horseradish peroxidase, a commonly used enzyme marker that can catalyze the production of color or light emission from substrates.

[0055] In this application, "ALP" refers to alkaline phosphatase, a commonly used enzyme label that catalyzes a colorimetric reaction of a substrate.

[0056] In this application, "CV value" refers to the coefficient of variation, which reflects the repeatability of the test results. The smaller the CV value, the better the repeatability.

[0057] In one aspect of this application, a "magnetic bead-small peptide-bispecific antibody" immunoassay system was constructed. This system involves coating magnetic beads with specific small peptides, and then conjugating anti-small peptide antibodies with specific antibodies against the analyte in vitro or through direct intracellular expression to create bifunctional antibodies (bispecific antibodies). The system utilizes the ability of the bispecific antibody to simultaneously bind to both the small peptides on the magnetic beads and the analyte in the sample, followed by a sandwich assay for detection. This system achieves highly efficient immobilization of the bispecific antibody through the highly specific binding of the small peptide to the anti-small peptide antibody. Compared to direct antibody coating or biotin-streptavidin bridging methods, this significantly improves antibody immobilization density and activity retention. The targeted binding effect of the bispecific antibody amplifies the detection signal while avoiding competitive interference from endogenous biotin, thus achieving highly sensitive and interference-resistant immunoassay.

[0058] In a first aspect of this application, an immunoassay kit is provided, comprising a small peptide-coated solid-phase carrier, a bispecific antibody, and a marker-labeled detection antibody, wherein the bispecific antibody comprises an anti-small peptide antibody and a linked capture antibody, the anti-small peptide antibody specifically binding to the small peptide, and both the capture antibody and the marker-labeled detection antibody specifically binding to the target and having different antigen recognition epitopes.

[0059] In some embodiments, the small peptide comprises 5 to 20 amino acid residues.

[0060] It should be noted that small peptides have low molecular weight and simple structure, enabling high-density and highly uniform coating on solid-phase support surfaces. Their low immunogenicity effectively reduces non-specific binding background in the detection system. Due to their small molecular weight and low steric hindrance, they facilitate efficient recognition and binding of anti-small peptide antibodies in bispecific antibodies, thereby achieving efficient and stable immobilization of bispecific antibodies on solid-phase supports, thus improving the sensitivity, stability, and anti-interference ability of the detection. Furthermore, small peptides can be efficiently prepared using solid-phase synthesis methods, resulting in high purity and good batch-to-batch consistency, facilitating large-scale production and quality control.

[0061] In some embodiments, the concentration of the small peptide on the peptide-coated magnetic beads is 2 mg / mL to 3 mg / mL. Non-limitingly, the concentration of the small peptide on the peptide-coated magnetic beads can be, but is not limited to, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, or any value or range between two of the above.

[0062] It should be noted that the small peptides within the above concentration range are beneficial for forming a stable and moderately dense small peptide coating layer on the surface of magnetic beads. This concentration range provides sufficient binding sites for the anti-small peptide antibodies in the bispecific antibody to recognize and bind, thereby improving the immobilization efficiency and detection sensitivity of the bispecific antibody; while avoiding insufficient coating and signal weakening due to too low a small peptide concentration, or aggregation between small peptide molecules or increased steric hindrance due to too high a concentration, which would affect the binding and detection stability of the bispecific antibody.

[0063] In some implementations, the solid support may be a conventional solid support in the art, such as, but not limited to, magnetic beads.

[0064] In some embodiments, the particle size of the magnetic beads is 1 μm to 5 μm. Non-limitingly, the particle size of the magnetic beads can be 1 μm, 2 μm, 3 μm, 4 μm, or 5 μm. Magnetic beads within this particle size range have suitable specific surface area and magnetic responsiveness. It is understood that magnetic beads of different sizes can be selected according to specific detection requirements.

[0065] In some embodiments, the markers in the marker-tagged detection antibodies can be of conventional types in the art to adapt to different detection platforms and signal readout methods. Exemplarily, the markers include, but are not limited to, one or more of enzyme markers, electrochemiluminescent markers, and fluorescent markers.

[0066] In some embodiments, the enzyme markers include, but are not limited to, at least one of horseradish peroxidase and alkaline phosphatase.

[0067] In some implementations, the electrochemiluminescent label includes, but is not limited to, ruthenium terpyridine.

[0068] In some embodiments, the fluorescent markers include, but are not limited to, at least one of FITC and Cy5.

[0069] In some embodiments, the above-described immunoassay kit further includes one or more of a detection substrate, buffer solution, blocking solution, positive control, and negative control.

[0070] In one aspect of this application, the core components of the immunoassay kit provided by this application include: small peptide-coated magnetic beads, anti-small peptide-anti-analyte bispecific antibody, labeled secondary antibody, detection substrate, and matching buffer, as detailed below:

[0071] 1. Small peptides: Select polypeptide sequences with small molecular weight (5-20 amino acid residues), low immunogenicity, and high specificity for binding to anti-small peptide antibodies (such as RGDSTE small peptide with sequence H2N-Arg-Gly-Asp-Ser-Thr-Glu-COOH, or KPQENY small peptide with sequence H2N-Lys-Pro-Gln-Glu-Asn-Tyr-COOH). These can be prepared by solid-phase synthesis with a purity ≥95%.

[0072] 2. Magnetic beads: Superparamagnetic microspheres with a particle size of 1-5 μm are selected, and the surface is covered with active groups of carboxyl, amino or epoxy groups for covalent binding of small peptides;

[0073] 3. Bispecific antibodies: Prepared through two methods:

[0074] In vitro conjugation method: Anti-small peptide monoclonal antibody and anti-analyte monoclonal antibody are covalently conjugated with a cross-linking agent (such as SMCC, SPDP), and then purified to obtain bifunctional antibody;

[0075] Intracellular direct expression method: Construct a recombinant expression vector containing the variable region gene of anti-small peptide antibody and the variable region gene of anti-detection antibody, transfect it into CHO or HEK293 cells, and obtain bispecific antibodies through cell culture and protein purification;

[0076] 4. Labeled secondary antibody: A specific antibody against another epitope of the analyte. The label can be horseradish peroxidase (HRP), alkaline phosphatase (ALP), or fluorescein (such as FITC, Cy5), or triple pyridine ruthenium.

[0077] 5. Detection of substrates: Select the corresponding substrate based on the label, such as TMB substrate for HRP, chemiluminescent substrate for luminol, pNPP substrate for ALP, etc.

[0078] 6. Supporting buffers: including magnetic bead washing buffer (PBS buffer containing 0.05% Tween-20, pH 7.4), incubation buffer (PBS buffer containing 1% BSA), and stop solution (2 mol / L H2SO4 or dedicated chemiluminescence stop solution).

[0079] In a second aspect of this application, a method for preparing the above-mentioned immunoassay kit is provided, comprising the following steps:

[0080] An immunoassay kit was prepared by providing a small peptide-coated solid-phase carrier, a bispecific antibody, and a labeled detection antibody.

[0081] In some implementations, the bispecific antibodies are prepared by in vitro conjugation or direct intracellular expression.

[0082] In some embodiments, the preparation method of the bispecific antibody includes covalently coupling an anti-small peptide antibody and a capture antibody using a cross-linking agent to prepare the bispecific antibody.

[0083] In some embodiments, the crosslinking agent includes one or more of SMCC (4-N-maleimide methylcyclohexane-1-carboxylic acid succinimide ester) and SPDP (N-succinimide-3-(2-pyridyl dithio)propionate).

[0084] In some embodiments, the concentration ratio of anti-small peptide antibody to capture antibody is (0.8~1.2):(0.8~1.2). Non-limitingly, the concentration ratio of anti-small peptide antibody to capture antibody can be, but is not limited to, 0.8:1, 0.8:1.2, 1:1, 1:0.8, 1.2:0.8, or any ratio or range between two of the above.

[0085] It should be noted that controlling the concentration ratio of anti-peptide antibody to capture antibody within the aforementioned range helps achieve a balanced binding of the two antibodies during the covalent coupling reaction. This avoids an excess of one antibody, resulting in an overly high proportion of that antibody and an insufficient proportion of the other in the formed bispecific antibody, thus affecting the bifunctional binding activity of the bispecific antibody. The prepared bispecific antibody can simultaneously maintain high affinity for the anti-peptide and efficient recognition ability of the target analyte, ensuring that it can be efficiently immobilized on the solid-phase support in the detection system while fully capturing the target analyte, thereby improving detection sensitivity and specificity.

[0086] In some embodiments, the conditions for covalent coupling include a temperature of 20°C to 30°C and a time of 0.5h to 1.5h. Non-limitingly, the temperature can be 20°C, 25°C, or 30°C; and the time can be 0.5h, 1h, or 1.5h.

[0087] In some embodiments, the preparation of bispecific antibodies includes constructing a recombinant expression vector containing a variable region gene for anti-small peptide antibodies and a variable region gene for capture antibodies, transfecting it into host cells for culture, and preparing bispecific antibodies.

[0088] In some embodiments, the host cell is an animal cell; further, the host cell includes, but is not limited to, CHO cells or HEK293 cells.

[0089] In some embodiments, the method for preparing a small peptide-coated solid-phase support includes mixing a small peptide and a solid-phase support and incubating them to prepare a small peptide-coated solid-phase support.

[0090] In some embodiments, the surface of the solid support is modified with active groups; further, the active groups include, but are not limited to, one or more of carboxyl, amino, and epoxy groups.

[0091] In some embodiments, the mass ratio of the small peptide to the solid support is (1~5):10. Without limitation, the mass ratio of the small peptide to the solid support can be, but is not limited to, 1:10, 2:10, 3:10, 4:10, 5:10, or any ratio or range between two of the above.

[0092] It should be noted that controlling the mass ratio of small peptides to the solid-phase support within the aforementioned range allows the small peptides to be distributed more uniformly on the surface of the solid-phase support. This facilitates efficient recognition and binding of the anti-small peptide antibodies, thereby improving the immobilization efficiency of the bispecific antibody and the stability and sensitivity of the detection system. If the proportion of small peptides is too low, it may result in insufficient binding sites, affecting the intensity of the detection signal; if the proportion is too high, it may cause mutual interference between small peptides or an overly dense coating layer, which is not conducive to the recognition and binding of the bispecific antibody.

[0093] In some embodiments, the incubation conditions include a temperature of 4°C to 37°C and a time of 8 hours to 16 hours. Non-limitingly, the incubation temperature can be 4°C, 10°C, 20°C, 30°C, or 37°C; the incubation time can be 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours.

[0094] In some embodiments, the preparation steps of small peptide-coated magnetic beads include:

[0095] 1. Activation of magnetic beads: Take 10 mg of carboxyl-modified magnetic beads, add 1 mL of MES buffer (0.1 mol / L, pH 5.0) to resuspend, add 50 μL of EDC solution (10 mg / mL) and 50 μL of NHS solution (10 mg / mL), and activate by shaking at room temperature for 30 min. After magnetic separation, wash twice with MES buffer.

[0096] 2. Small peptide coupling: The activated magnetic beads were resuspended in 1 mL of incubation buffer, 1-5 mg of synthetic small peptides were added, and the mixture was incubated at 4 °C with shaking for 12 h to allow the small peptides to form amide bonds with the amino groups on the surface of the magnetic beads through the carboxyl groups.

[0097] 3. Blocking: Discard unbound small peptides, add 1 mL of blocking solution containing 2% BSA, and block at room temperature with shaking for 2 h to block unreacted active sites on the surface of magnetic beads;

[0098] 4. Storage: After magnetic separation, wash three times with washing buffer, resuspend in PBS buffer containing 0.02% sodium azide, and store at 4°C for later use.

[0099] In a third aspect of this application, an immunoassay method is provided, comprising detecting a sample to be tested using the aforementioned immunoassay kit.

[0100] In some implementations, the immune detection method includes the following steps:

[0101] A solid-phase support coated with small peptides and a bispecific antibody were mixed and incubated to prepare a solid-phase support-small peptide-bispecific antibody complex.

[0102] The sample to be tested and the solid-phase carrier-small peptide-double antibody complex were mixed and incubated to prepare the solid-phase carrier-small peptide-double antibody-target complex.

[0103] A sandwich complex was prepared by mixing a solid-phase carrier-small peptide-biantibody-target complex and a marker-labeled detection antibody and incubating the mixture.

[0104] Add the detection substrate to initiate the reaction, and then detect the reaction signal.

[0105] In some embodiments, the concentration ratio of the bispecific antibody to the marker-labeled detection antibody is (1~5):1. Non-limitingly, the concentration ratio of the bispecific antibody to the marker-labeled detection antibody can be, but is not limited to, 1:1, 2:1, 3:1, 4:1, 5:1, or any ratio or range between two of the above.

[0106] It should be noted that the bispecific antibody can fully bind to the small peptides on the surface of the solid-phase carrier and effectively capture the target analyte in the test sample. At the same time, the detection antibody labeled by the label binds to another epitope of the target analyte to form a stable sandwich complex, which ensures sufficient signal intensity and avoids nonspecific background increase caused by excessive labeling antibody.

[0107] In some implementations, the sample to be tested includes plasma or serum.

[0108] In some implementations, the above-described immunoassay method is for non-diagnostic purposes. Exemplarily, it can be used for biopharmaceutical process control, environmental monitoring, or food safety testing.

[0109] In some implementations, the above-described immunoassay method is used for diagnostic purposes.

[0110] In some embodiments, the immunoassay kit provided in this application employs a modified sandwich detection method, the principle of which is as follows:

[0111] 1. Capture stage: Small peptide-coated magnetic beads are mixed and incubated with bispecific antibodies. The anti-small peptide antibody domain in the bispecific antibodies specifically binds to the small peptide on the surface of the magnetic beads, forming a "magnetic bead-small peptide-bispecific antibody" complex, thereby achieving efficient immobilization of the bispecific antibodies on the surface of the magnetic beads.

[0112] 2. Binding stage: The serum sample to be tested is added to the complex. The anti-analyte antibody domain in the bispecific antibody specifically binds to the analyte in the sample, forming a "magnetic bead-small peptide-bispecific antibody-analyte" complex.

[0113] 3. Signal amplification stage: Add labeled secondary antibody. The labeled secondary antibody binds to another epitope of the analyte to form a complete sandwich complex. At this time, the targeting binding effect of the two antibodies causes more labeled molecules to accumulate on the surface of the magnetic beads, thereby amplifying the signal.

[0114] 4. Detection stage: The corresponding substrate is added, and the label catalyzes the reaction of the substrate. The reaction signal (absorbance, luminescence intensity or fluorescence intensity) is detected by equipment such as an enzyme-linked immunosorbent assay (ELISA) reader or chemiluminescence detector. The signal intensity is positively correlated with the concentration of the analyte, thus achieving quantitative detection.

[0115] In a fourth aspect of this application, the above-described immunoassay kit is provided for use in the preparation of products for detecting tumor markers or myocardial markers.

[0116] In some implementations, tumor markers include, but are not limited to, carcinoembryonic antigen (CEA).

[0117] In some implementations, cardiac markers include, but are not limited to, cardiac troponin I.

[0118] In another aspect of this application, the principle by which the technical solution provided in the embodiments of this application solves the technical problem includes:

[0119] 1. Enhanced Sensitivity: The binding specificity and affinity between small peptides and anti-small peptide antibodies are high, enabling high-density immobilization of bispecific antibodies on the surface of magnetic beads. Compared with direct antibody coating, the immobilization efficiency is improved by more than 30%. At the same time, the bifunctional structure of the bispecific antibodies makes the binding sites of the analyte more concentrated. Combined with the signal superposition effect of the labeled secondary antibody, it significantly enhances the signal response of low concentrations of analytes, improving the sensitivity by 1-2 orders of magnitude.

[0120] 2. Elimination of biotin interference: This system does not require biotin-streptavidin bridging, completely avoiding the competitive binding problem of endogenous biotin in serum, and the detection results are not affected by biotin concentration;

[0121] 3. Improve antibody utilization: Small peptide modification does not affect the antibody active site. The bispecific antibody is immobilized through specific binding, avoiding the problem of the antibody active site being blocked when directly coated. The antibody utilization is increased by more than 40%, reducing the detection cost.

[0122] 4. Enhanced stability: The binding stability of small peptides to anti-small peptide antibodies is better than that of direct antibody coating and biotin-streptavidin binding. It remains stable over a wide temperature range (4-37℃) and pH range (6.0-8.0), with a detection repeatability coefficient of variation (CV) ≤5%.

[0123] The following are some examples.

[0124] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0125] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0126] In the following examples, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-RGDSTE monoclonal antibody, as well as the nucleic acid sequences encoding the aforementioned variable regions, are as follows:

[0127] The amino acid sequence of the heavy chain variable region of the anti-RGDSTE monoclonal antibody is SEQ ID NO: 3.

[0128] EVKLVESGGGLMTPGGSLKFSCVASGFTFNTYAMSWVRQTPEKRLEWVASINTGGATFYADSVKGRFTISRDNARNILYLHMSSLRSEDTAMYFCTREGDWYGPFAYWGQGTLVTVSS

[0129] The amino acid sequence of the light chain variable region of the anti-RGDSTE monoclonal antibody is SEQ ID NO: 4.

[0130] DIVMTQSQRFMSTSVGDRVSVTCKASQNVGVNVAWYQQTPGHSPKALIYSASYHYSGVPDRFAGSGSGTDFTLTIDNVQSEDLADYFCQQYNSFPFTFGSGTKLEMK

[0131] The nucleic acid sequence encoding the variable region of the heavy chain of the anti-RGDSTE monoclonal antibody is SEQ ID NO: 5.

[0132] GAGGTGAAGCTGGTGGAATCTGGAGGCGGCCTGATGACACCTGGCGGCTCTCTGAAGTTCAGCTGTGTGGCCTCTGGCTTCACCTTTAATACCTATGCTATGTCTTGGGTCAGACAGACCCCTGAGAAGCGGCTGGAATGGGTGGCTTCCATCAACACCGGCGGAGCTACCTTCTAC GCCGATAGCGTGAAAGGCAGATTCACCATCAGCCGGGACAACGCCAGAAACATCCTGTAACCTGCACATGAGCAGCCTGAGAAGCGAGGACACCGCCATGTACTTCTGCACAAGGGAGGGTGACTGGTACGGCCCCTTCGCCTACTGGGGCCAGGGCACACTGGTGACCGTGTCCTCC

[0133] The nucleic acid sequence encoding the light chain variable region of the anti-RGDSTE monoclonal antibody is SEQ ID NO: 6.

[0134] GATATCGTGATGACCCAGAGCCAGAGATTCATGAGCACGTCTGTGGGCGACAGAGTGTCCGTGACCTGTAAGGCCAGCCAGAACGTCGGCGTGAACGTGGCCTGGTACCAGCAAACACCTGGGCACAGCCCCAAGGCTCTGATCTACTCCGCCAGCTACC ACTACAGCGGCGTGCCTGATCGGTTCGCCGGCTCCGGCTCTGGAACCGACTTCACCCTGACCATCGACAATGTGCAGTCCGAGGACCTGGCCGACTACTTCTGCCAGCAGTACAACAGCTTTCCTTTCACCTTCGGCTCTGGCACCAAGCTGGAAATGAAG

[0135] In the following examples, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-CEA monoclonal antibody, as well as the nucleic acid sequences encoding the aforementioned variable regions, are as follows:

[0136] The amino acid sequence of the variable region of the heavy chain of the anti-CEA monoclonal antibody is SEQ ID NO: 7.

[0137] EVKLQESGPELKKPGETVKLSCKASGYTFSNYGMNWVEQAPGKGLRWMGWINTNTGDPTYAEEFKGSFAFSLETSASTAYLQVNSLTDEDTATYFCARGTGTTAYWGQGTLVTVSA

[0138] The nucleic acid sequence encoding the variable region of the heavy chain of the anti-CEA monoclonal antibody is SEQ ID NO: 8.

[0139] GAGGTGAAGCTGCAGGAGAGCGGACCTGAGCTGAAGAAGCCCGGCGAGACAGTGAAACTGAGCTGTAAAGCCAGCGGCTACACCTTCTCCAACTACGGCATGAACTGGGTGGAACAGGCCCCTGGCAAGGGCCTGCGGTGGATGGGCTGGATCAACACCAATACCGGAGATCCA ACCTACGCCGAGGAATTCAAGGGCAGCTTCGCCTTTTCTCTGGAAACCAGCGCCTCTACCGCCTACCTGCAAGTGAACAGCCTCACCGACGAGGACACCGCTACATACTTCTGCGCCAGAGGCACAGGAACAACAGCTTATTGGGGCCAGGGCACCCTGGTCACCGTGTCCGCC

[0140] Amino acid sequence of the variable region of the light chain of the anti-CEA monoclonal antibody SEQ ID NO: 9:

[0141] DILMTQSPASLSADVGRTVTITCRASENLYSYLAWYEEKQGNSPQLLVYNAATLAEGVPARFSGRGSGTAFSLKINSLQPEDFGTYYCQHHNVSPYTFGGGTKLEIK

[0142] The nucleic acid sequence encoding the variable region of the light chain of the above-mentioned anti-CEA monoclonal antibody is SEQ ID NO: 10:

[0143] GATATCCTGATGACACAGAGCCCTGCTTCTCTGAGCGCCGACGTGGGCCGGACCGTGACCATCACCTGTAGAGCCTCTGAGAACCTGTACAGCTACCTGGCCTGGTATGAGGAAAAACAGGGCAATAGCCCCCAGCTGCTGGTCTACAACGCCGCTACAC TGGCCGAGGGCGTGCCTGCCAGATTCAGCGGAAGAGGCAGCGGCACCGCCTTTTAGCCTCAAGATCAACTCCCTGCAACCTGAGGACTTCGGCACCTACTACTGCCAGCACCACAACGTGTCCCCATACACCTTCGGAGGCGGCACAAAGCTGGAAATCAAG

[0144] In the following examples, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-KPQENY antibody, as well as the nucleic acid sequences encoding the aforementioned variable regions, are as follows:

[0145] Amino acid sequence of the variable region of the heavy chain of the anti-KPQENY antibody SEQ ID NO: 11:

[0146] QVQLKESGPGLVAPSQSLSITCTVSGFSLSSYGVSWIRQPPGKGLEWLGVIWGDGSTNYHSALISRLSITKDNSKSQVFLKLNSLQTDDTATYYCVGFAYWGQGTLVTVSA

[0147] The nucleic acid sequence encoding the variable region of the heavy chain of the above-mentioned anti-KPQENY antibody is SEQ ID NO: 12:

[0148] CAAGTGCAGCTGAAAGAGAGTGGGCCCGGACTCGTGGCCCCTTCTCAGTCTCTGAGCATCACCTGTACTGTGTCCGGCTTTTCTCTGAGCAGCTATGGCGTGTCTTGGATCCGGCAGCCTCCTGGCAAGGGTCTGGAATGGCTGGGCGTGATCTGGGGCGATGGCAGCACCAACTACCACTCCGCTCTGATCAGCAGACTGTCCATCACCAAGGACAATTCCAAGAGCCAGGTCTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACCGCTACCTACTACTGCGTGGGCTTCGCCTACTGGGGCCAGGGCACACTGGTCACAGTGTCCGCC

[0149] Amino acid sequence of the light chain variable region of the anti-KPQENY antibody, SEQ ID NO: 13:

[0150] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPERLIYLVSKLESGVPDRFTGSGSGTDFTLKISRVEAEDLGVYHCWQGTHFPYTFGGGTKLEIL

[0151] Nucleic acid sequence encoding the light chain variable region of the anti-KPQENY antibody, SEQ ID NO: 14:

[0152] GATGTGGTGATGACCCAGACCCCTCTGACCCTGAGCGTGACAATCGGCCAGCCTGCTAGCATCAGCTGCAAGTCCTCCCAGAGCCTGCTGGACAGCGATGGCAAGACCTACCTGAACTGGCTGTTCCAGAGACCTGGACAATCTCCTGAGCGGCTGATCTACCTGGTGTCCAAGCTGGAATCCGGCGTGCCCGACAGATTTACAGGGTCTGGCAGCGGAACCGACTTCACCCTGAAGATCTCCAGAGTCGAAGCCGAGGACCTGGGCGTGTACCACTGTTGGCAGGGCACCCACTTCCCCTACACCTTCGGCGGCGGCACAAAACTGGAGATCCTG

[0153] In the following examples, the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-CTNI antibody, as well as the nucleic acid sequences encoding the aforementioned variable regions, are as follows:

[0154] Amino acid sequence of the variable region of the heavy chain of anti-CTNI antibody SEQ ID NO: 15:

[0155] QVQLQESGPSLVKPSQTLSLTCTVSGFSLANHAVDWVRQAPGKAPEWLGGIDDVGGTAYNPALKSRLSISRDTSRSQVSLSLSSVSTEDTAVYHCYASGTKYPFNLMSADEDWGPGLLVTVSS

[0156] The nucleic acid sequence encoding the variable region of the heavy chain of the above-mentioned anti-CTNI antibody is SEQ ID NO: 16:

[0157] CAAGTGCAGCTGCAGGAGAGCGGCCCCAGCCTGGTGAAGCCTAGCCAGACCCTGAGCCTCACCTGTACCGTGTCCGGCTTCAGCCTGGCCAACCACGCCGTGGACTGGGTCAGACAGGCCCCTGGAAAAGCCCCAGAATGGCTGGGCGGAATCGACGACGTGGGCGGCACAGCTTATAATCCTG CCCTGAAGAGCAGACTGTCCATCAGCCGGGACACAAGCAGATCTCAGGTGTCTCTGAGCCTGAGCTCTGTGTCCACCGAAGATACAGCTGTGTACCACTGCTACGCCAGCGGCACCAAGTACCCCTTCAACCTGATGAGCGCCGACGAGGATTGGGGCCCTGGCCTGCTGGTTACCGTGAGCTCT

[0158] Amino acid sequence of the variable region of the light chain of anti-CTNI antibody SEQ ID NO: 17:

[0159] YELTQPTSVSVALGQTAKITCQGDLLDEKYTAWYQQKPGQAPLKVIYRDSERPSGILDRFSGSSSGKTATLIISGARTEDEADYYCLSVDNHHIPFFGSGTRLTVL

[0160] The nucleic acid sequence encoding the variable region of the light chain of the above-mentioned anti-CTNI antibody is SEQ ID NO: 18:

[0161] TACGAGCTGACCCAACCTACAAGCGTGTCCGTCGCCCTGGGCCAGACCGCCAAGATCACCTGCCAGGGCGACCTGCTGGACGAAGTATACAGCTTGGTACCAGCAGAAGCCCGGCCAAGCTCCTCTGAAGGTGATCTACCGGGACAGCGAACGGCCT AGCGGCATCCTGGATAGATTCAGCGGCAGCAGCAGCGGCAAGACCGCCACCCTGATCATCAGCGGCGCCAGAACCGAAGATGAGGCCGATTACTACTGCCTGAGCGTCGACAACCACCACATCCCCTTCTTCGGCTCCGGAACAAGACTGACCGTGCTG

[0162] Example 1: Preparation of a tumor marker CEA detection kit based on in vitro conjugated bispecific antibodies

[0163] 1. Preparation of small peptides: RGDSTE (SEQ ID NO: 1) small peptide (sequence H2N-Arg-Gly-Asp-Ser-Thr-Glu-COOH) was synthesized by solid-phase synthesis and purified by HPLC with a purity of 98.2%.

[0164] 2. Preparation of small peptide-coated magnetic beads:

[0165] Magnetic bead activation: Take 10 mg of carboxyl magnetic beads (particle size 2 μm), resuspend in MES buffer, add EDC and NHS solution and activate for 30 min;

[0166] Conjugation: Add 2 mg of RGDSTE peptide, incubate at 4°C for 16 h, separate by magnetic adsorption, and wash away unbound peptides;

[0167] Sealing: Seale with 2% BSA for 2 hours, wash and store at 4°C;

[0168] 3. Preparation of bispecific antibodies:

[0169] Take 1 mL each of anti-RGDSTE monoclonal antibody (concentration 1 mg / mL) and anti-CEA monoclonal antibody (concentration 1 mg / mL), add 50 μL of SMCC crosslinking agent, and react at room temperature for 1 h;

[0170] Free cross-linking agent was removed by dialysis, and the anti-RGDSTE-anti-CEA bispecific antibody was obtained by gel filtration chromatography at a concentration of 0.8 mg / mL.

[0171] 4. Kit assembly: Package the small peptide-coated magnetic beads, double antibody, HRP-labeled anti-CEA secondary antibody, TMB substrate, washing buffer, blocking solution, and CEA standard in the specified proportions to prepare the kit.

[0172] Example 2: Preparation of a cTnI detection kit based on intracellular expression of bispecific antibodies for myocardial markers

[0173] 1. Preparation of small peptides: KPQENY (SEQ ID NO: 2) small peptides (sequence H2N-Lys-Pro-Gln-Glu-Asn-Tyr-COOH) were synthesized and purified by HPLC with a purity of 97.8%;

[0174] 2. Preparation of small peptide-coated magnetic beads:

[0175] Magnetic bead activation: Take 10 mg of amino magnetic beads (particle size 3 μm), resuspend them in PBS buffer (pH 7.4), add epoxy activator, and activate at room temperature for 40 min;

[0176] Conjugation: Add 3 mg of KPQENY peptide and incubate at 37°C for 8 hours to form a covalent bond;

[0177] Blocking: Block with 1% ovalbumin for 1.5 hours, wash and store at 4°C;

[0178] 3. Preparation of bispecific antibodies:

[0179] Construction of recombinant vectors: The variable region genes of anti-KPQENY antibody and anti-cTnI antibody were inserted into the pCDNA3.1 expression vector;

[0180] Cellular expression: CHO-K1 cells were transfected and cultured in serum-free medium for 72 h, and the supernatant was collected.

[0181] Purification: The anti-KPQENY-anti-cTnI bispecific antibody was obtained by Protein A affinity chromatography at a concentration of 0.6 mg / mL.

[0182] 4. Kit assembly: Package the small peptide-coated magnetic beads, cell expression double antibody, ALP-labeled anti-cTnI secondary antibody, pNPP substrate, matching buffer and cTnI standard to prepare the kit.

[0183] Example 3: Effect of different small peptide concentrations on detection results

[0184] 1. Experimental design: Coated magnetic beads with small peptide concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL were prepared respectively, with other preparation conditions the same as in Example 1, and used for CEA detection;

[0185] 2. Detection results: When the concentration of small peptides is 2-3 mg / mL, the small peptide coating density on the magnetic bead surface is suitable, the double antibody immobilization efficiency is the highest, the detection signal intensity is the largest, and the CV value is the smallest (≤3%). When the concentration is below 2 mg / mL, the coating density is insufficient and the signal is weak. When the concentration is above 3 mg / mL, the small peptides are prone to polymerization, and the signal stability decreases.

[0186] Comparative Example 1: CEA detection kit with directly coated antibodies

[0187] 1. Preparation: Take 10 mg of carboxyl magnetic beads, directly couple them with 1 mg / mL anti-CEA antibody, and block them to prepare antibody-coated magnetic beads;

[0188] 2. Detection procedure: Following the conventional sandwich method, the antibody-coated magnetic beads are incubated sequentially with the sample and HRP-labeled secondary antibody, and the CEA standard is detected.

[0189] 3. Drawbacks: Low antibody fixation efficiency, weak detection signal at low concentrations of CEA (1 ng / mL), sensitivity of 0.5 ng / mL, and CV value of 8.6% for serum sample detection.

[0190] Comparative Example 2: Biotin-Streptavidin-bridged CEA detection kit

[0191] 1. Preparation: Magnetic beads were modified with streptavidin and then bound to biotinylated anti-CEA antibody to prepare bridging magnetic beads;

[0192] 2. Detection procedure: Conventional sandwich method detection, with interference groups containing different concentrations of biotin (0-100 ng / mL);

[0193] 3. Defects: The sensitivity is 0.3 ng / mL when there is no biotin interference. When the biotin concentration is ≥50 ng / mL, the detection signal drops by 40%, resulting in false negative results. It has poor anti-interference ability.

[0194] Experimental results

[0195] (1) Sensitivity comparison experiment

[0196] The results of testing CEA standards (concentrations of 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL) are shown in Table 1 below:

[0197] Table 1

[0198]

[0199] Conclusion: The detection limit of Example 1 was significantly lower than that of Comparative Example 1 and Comparative Example 2, and the sensitivity was improved by 10-25 times.

[0200] (2) Biotin interference experiment

[0201] Different concentrations of biotin (0, 20, 50, 100, 200 ng / mL) were added to 1 ng / mL CEA standard, and the inhibition rates of the detection signal are shown in Table 2 below:

[0202] Table 2

[0203]

[0204] Conclusion: The signal inhibition rate of Example 1 was consistently ≤2.3%, and was basically unaffected by biotin; the inhibition rate of Comparative Example 2 increased significantly when the biotin concentration was ≥50 ng / mL, indicating poor anti-interference ability.

[0205] (3) Comparative experiment of serum sample detection

[0206] Twenty clinical serum samples (CEA concentration 0.1-50 ng / mL) were selected and tested using the kits from Example 1, Comparative Example 1, and Comparative Example 2, respectively. The results are shown in Table 3 below:

[0207] Table 3

[0208]

[0209] Conclusion: The repeatability and accuracy of Example 1 are significantly better than those of the comparative example, and there are no false negative results, making it more suitable for clinical serum sample testing.

[0210] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0211] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An immunoassay kit, characterized by comprising: It includes a small peptide-coated solid-phase carrier, a bispecific antibody, and a marker-labeled detection antibody, wherein the bispecific antibody includes an anti-small peptide antibody and a linked capture antibody, the anti-small peptide antibody specifically binding to the small peptide, and both the capture antibody and the marker-labeled detection antibody specifically binding to the target and having different antigen recognition epitopes.

2. The immunoassay kit of claim 1, wherein The small peptide comprises 5 to 20 amino acid residues; Optionally, the amino acid sequence of the small peptide is as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. The immunoassay kit of claim 2, wherein The solid support includes magnetic beads; optionally, the particle size of the magnetic beads is 1μm to 5μm.

4. The immunoassay kit according to any one of claims 1 to 3, wherein One or more of the following conditions must be met: (1) The markers include one or more of enzyme markers, electrochemiluminescent markers, and fluorescent markers; Optionally, the enzyme marker includes at least one of horseradish peroxidase and alkaline phosphatase; Optionally, the electrochemiluminescent label includes ruthenium terpyridine; Optionally, the fluorescent marker includes at least one of FITC and Cy5; (2) The immunoassay kit also includes one or more of the following: detection substrate, buffer solution, blocking solution, positive control and negative control.

5. A method for preparing an immunoassay kit according to any one of claims 1 to 4, characterized in that, It includes the following steps: The immunoassay kit is prepared by providing a solid-phase carrier coated with the small peptide, the bispecific antibody, and the marker-labeled detection antibody, respectively.

6. The method for preparing an immunochromatographic test kit according to claim 5, wherein the antibody is an antibody against the antigen. The method for preparing the bispecific antibody includes covalently coupling the anti-small peptide antibody and the capture antibody using a cross-linking agent to prepare the bispecific antibody; Optionally, one or more of the following conditions must be met: The crosslinking agent includes one or more of SMCC and SPDP; The concentration ratio of the anti-small peptide antibody to the capture antibody is (0.8~1.2):(0.8~1.2). The conditions for covalent coupling include: a temperature of 20℃~30℃ and a time of 0.5h~1.5h.

7. The method for preparing an immunochromatographic test kit according to claim 5, wherein the antibody is an antibody against the antigen. The method for preparing the bispecific antibody includes constructing a recombinant expression vector containing a variable region gene for an anti-small peptide antibody and a variable region gene for a capture antibody, transfecting it into host cells for culture, and preparing the bispecific antibody. Optionally, the host cells include one or more of CHO cells and HEK293 cells.

8. The method for preparing the immunoassay kit according to any one of claims 5 to 7, characterized in that, The method for preparing the small peptide-coated solid-phase support includes mixing the small peptide and the solid-phase support and incubating them to prepare the small peptide-coated solid-phase support. Optionally, one or more of the following conditions must be met: The surface of the solid support is modified with active groups, which include one or more of carboxyl, amino and epoxy groups. The mass ratio of the small peptide to the solid support is (1~5):10; The incubation conditions include a temperature of 4℃ to 37℃ and a time of 8h to 16h.

9. An immunoassay method characterized in that, It includes using the immunoassay kit according to any one of claims 1 to 4 to detect the target analyte in the sample to be tested; Optionally, the immune detection method includes the following steps: A solid-phase support coated with small peptides and a bispecific antibody were mixed and incubated to prepare a solid-phase support-small peptide-bispecific antibody complex. The sample to be tested and the solid-phase carrier-small peptide-double antibody complex were mixed and incubated to prepare the solid-phase carrier-small peptide-double antibody-target complex. The solid-phase carrier-small peptide-biantibody-target complex and the label-labeled detection antibody are mixed and incubated to prepare a sandwich complex; Add the detection substrate to initiate a reaction, and then detect the reaction signal. Optionally, the concentration ratio of the bispecific antibody to the marker-labeled detection antibody is (1~5):1; The sample to be tested includes plasma or serum.

10. The use of the immunoassay kit according to any one of claims 1 to 4 in the preparation of products for detecting tumor markers or myocardial markers.

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