Kit and method for measuring cytotoxin-associated protein a

Abstract

The embodiment of the application belongs to the technical field of detection, and relates to a kit and a method for measuring cytotoxin-related protein A, comprising Ra reagent, Rb reagent, Rc reagent and a luminescent substrate; wherein the Ra reagent comprises streptavidin-modified magnetic microparticles; the Rb reagent comprises a biotin-labeled cytotoxin-related protein A antibody; and the Rc reagent comprises a chemiluminescent substance-labeled cytotoxin-related protein A antibody. The application has high sensitivity and accurate test results.

Description

Technical Field

[0001] This application relates to the field of detection technology, and in particular to kits and methods for determining cytotoxic-associated protein A. Background Technology

[0002] Helicobacter pylori (HP) is a spiral-shaped, microaerophilic, Gram-negative bacterium. The International Agency for Research on Cancer (IARC) of the World Health Organization has classified HP as a Group 1 carcinogen. Based on the presence or absence of cytotoxin-associated gene A (CagA) expression, HP strains can be divided into two types: Type I, a highly virulent strain containing the CagA gene and expressing cellular CagA protein; and Type II, a low-virulence strain lacking the CagA gene and not expressing CagA protein. Cytotoxin-associated gene A is an important virulence factor encoded by HP. As the only oncoprotein derived from bacteria, once it enters the cell, it can interact with multiple molecules, disrupting normal cellular signaling pathways and causing cytopathic effects and transformation. Studies have shown that cytotoxin-associated gene A not only promotes tumor development and progression in vivo by activating signaling pathways such as SHP-2, Grb2, and β-catenin, but is also closely related to the occurrence of cardiovascular and cerebrovascular diseases such as coronary heart disease and atherosclerosis.

[0003] Currently, common methods for detecting Helicobacter pylori include breath tests, fecal HP antigen detection, and serum antibody detection. However, these methods cannot differentiate between different strains of the infecting bacteria. The most widely used method for detecting cytotoxin-associated protein A (CPA) is enzyme-linked immunosorbent assay (ELISA). ELISA is an enzyme-labeled solid-phase immunoassay technique. Its basic principle involves binding antigens (or antibodies) to a solid-phase carrier, and then sequentially coating the antigen (or antibody) with bovine serum albumin (BSAB) onto the microwells of the carrier to form a composite layer. During detection, the analyte and biotinylated antigen (or antibody) bind to the ELISA plate according to a specific program. The information is then amplified by avidinized horseradish peroxidase (HRP), and the detection is completed using 3,3',5,5'-tetramethylbenzidine (TMB) and sulfuric acid as the substrate and stop solution, respectively. The disadvantages of this method are: when using an ELISA microplate as the solid-phase carrier, the analyte on the carrier surface cannot fully contact the analyte in the sample, resulting in low reaction efficiency and a long reaction time. Furthermore, the specificity and sensitivity of ELISA need improvement. Summary of the Invention

[0004] The purpose of this application is to provide a kit and method for determining cytotoxin-associated protein A. This application has high sensitivity and accurate test results.

[0005] To address the aforementioned technical problems, this application provides a kit for determining cytotoxin-associated protein A, employing the following technical solution:

[0006] A kit for determining cytotoxic protein A, comprising: Ra reagent, Rb reagent, Rc reagent and luminescent substrate;

[0007] The Ra reagent includes streptavidin-modified magnetic microparticles;

[0008] The Rb reagent includes a biotin-labeled antibody against cytotoxic protein A;

[0009] The Rc reagent includes a chemiluminescent antibody labeled with cytotoxic-associated protein A.

[0010] Furthermore, the kit also includes cleaning solution, calibrators, and quality control materials;

[0011] The cleaning solution includes 15.76 mg / mL Tris-HCl, 0.5 mg / mL Proclin 300, and 0.001 mg / mL Tween-20.

[0012] Furthermore, the luminescent substrate comprises 0.1 mg / mL of a 1,2-dioxane derivative.

[0013] Furthermore, the concentration of the streptavidin-modified magnetic microparticles is ≥0.3 mg / mL;

[0014] The concentration of the biotin-labeled cytotoxic protein A antibody is ≥10 μg / mL;

[0015] The concentration of the chemiluminescent material-labeled cytotoxin-associated protein A antibody is ≥5 μg / mL.

[0016] Furthermore, the biotin includes N-hydroxysuccinimide biotin;

[0017] And / or, the chemiluminescent material includes alkaline phosphatase.

[0018] Furthermore, the streptavidin-modified magnetic microparticles have a particle size of 1–3 μm.

[0019] Furthermore, the streptavidin-modified magnetic microparticles are prepared via the following steps:

[0020] After mixing the magnetic microparticles with a vortex mixer, morpholine ethanesulfonic acid was added to enrich the magnetic microparticles, and the supernatant was removed.

[0021] Morpholin ethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and streptavidin were added sequentially, and the mixture was rotated to mix and react.

[0022] After the reaction is complete, place the mixture on a magnetic rack, discard the supernatant, add the blocking liquid, and rotate to mix the reaction to obtain the streptavidin-modified magnetic microparticles.

[0023] Furthermore, the biotin-labeled cytotoxin-associated protein A antibody is prepared via the following steps:

[0024] Add the cytotoxin-associated protein A antibody to PBS buffer, mix well, then add activated biotin and rotate at room temperature in the dark.

[0025] After the reaction was complete, the sample was transferred to a cellulose dialysis bag and dialyzed with PBS buffer to obtain the biotin-labeled cytotoxin-associated protein A antibody.

[0026] Furthermore, the chemiluminescent material-labeled cytotoxin-associated protein A antibody is prepared via the following steps:

[0027] Take a chemiluminescent material, add 2-iminothionane hydrochloride solution to it, mix well and react at room temperature, then add glycine solution to react and obtain an activated chemiluminescent material.

[0028] Take the cytotoxin-associated protein A antibody, add a solution of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester sodium salt, vortex mix and react at room temperature, add glycine solution and react to obtain the activated antibody.

[0029] The activated antibody and the activated chemiluminescent substance were vortexed and mixed, reacted at room temperature, N-ethylmaleimide was added, vortexed and mixed, reacted at room temperature, ethanolamine was added, vortexed and mixed, reacted at room temperature, and then desalted and purified to obtain the purified chemiluminescent substance-labeled cytotoxic protein A antibody.

[0030] To address the aforementioned technical problems, this application also provides a method for determining cytotoxin-associated protein A, employing the following technical solution:

[0031] A method for determining cytotoxin-associated protein A, using the kit described above.

[0032] Compared with the prior art, the embodiments of this application have the following main advantages:

[0033] The kit described in this application uses a fully automated chemiluminescence immunoassay analyzer as the detection tool, employing chemiluminescence to detect cytotoxin-associated protein A. It offers rapid detection, simple operation, and good reproducibility. The kit utilizes a binding system of streptavidin magnetic microparticles and biotin-labeled derivatives to reduce non-specific adsorption and improve sample accuracy. Simultaneously, it avoids magnetic microparticle aggregation caused by direct antibody coating, thus resolving stability issues. Compared with existing technologies, the kit presented in this application offers advantages such as rapid testing, good reproducibility, stable reagent performance, high sensitivity, and accurate results. Attached Figure Description

[0034] To more clearly illustrate the solutions in this application, the accompanying drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a flowchart of an embodiment of the method for determining cytotoxin-associated protein A according to this application. Detailed Implementation

[0036] 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 pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application, are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0037] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0038] The following embodiments are provided to facilitate a better understanding of this application, but do not limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0039] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0040] This application provides a kit for the determination of cytotoxic protein A (CagA) by magnetic microparticle chemiluminescence assay. The kit includes Ra reagent, Rb reagent, and Rc reagent. The Ra reagent includes streptavidin-modified magnetic microparticles; the Rb reagent includes biotin-labeled cytotoxic protein A antibody; and the Rc reagent includes chemiluminescently labeled cytotoxic protein A antibody.

[0041] As an option in this application, the luminescent substrate includes 0.1 mg / mL of a 1,2-dioxane derivative.

[0042] The kit also includes a washing solution, calibrators, and quality control samples. The washing solution comprises 15.76 mg / mL Tris-HCl, 0.5 mg / mL Proclin 300, and 0.001 mg / mL Tween-20. In the washing solution, Tris-HCl serves as a buffer, Proclin 300 as a preservative, and Tween-20 as a surfactant. The calibrators in this application contain 100–1000 pg / mL of cytotoxic protein A antibody; the quality control samples contain 10–1000 pg / mL of cytotoxic protein A antibody.

[0043] In this embodiment, the cleaning solution is prepared by the following steps: 1.576g of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 0.0001g of Tween-20 and 0.05g of Proclin 300 are dissolved in 80mL of purified water, stirred and mixed, and then the volume is adjusted to 100mL to obtain the cleaning solution for later use.

[0044] Optional to this application, the concentration of the streptavidin-modified magnetic microparticles is ≥0.3 mg / mL; the concentration of the biotin-labeled cytotoxic-associated protein A antibody is ≥10 μg / mL; and the concentration of the chemiluminescent substance-labeled cytotoxic-associated protein A antibody is ≥5 μg / mL.

[0045] Optionally, the streptavidin-modified magnetic microparticles may have a particle size range of 1–3 μm. For example, 1 μm, 2.5 μm, or 3 μm.

[0046] Optionally, the chemiluminescent material may include alkaline phosphatase; and / or the biotin may include N-hydroxysuccinimide biotin (NHS-Biotin).

[0047] The chemical structure of the N-hydroxysuccinimide biotin is as follows:

[0048]

[0049] The chemical structure of the alkaline phosphatase is as follows:

[0050]

[0051] As an optional provision of this application, in the chemiluminescent material-labeled cytotoxin-associated protein A antibody, the molar ratio of the chemiluminescent material to the cytotoxin-associated protein A antibody (monoclonal antibody) is (1-10):1; in the biotin-labeled cytotoxin-associated protein A antibody, the molar ratio of biotin to the cytotoxin-associated protein A antibody (monoclonal antibody) is (1-20):1.

[0052] Furthermore, the streptavidin-modified magnetic microparticles of this application are prepared by the following steps:

[0053] After mixing the magnetic microparticles (i.e., magnetic beads) with a vortex mixer, morpholine ethanesulfonic acid was added to enrich the magnetic microparticles, and the supernatant was removed.

[0054] Morpholin ethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and streptavidin were added sequentially, and the mixture was rotated to carry out the coupling reaction.

[0055] After the coupling reaction is complete, the mixture is placed on a magnetic rack, the supernatant is discarded, a blocking solution is added, and the mixture is rotated to mix and obtain the streptavidin-modified magnetic microparticles.

[0056] In this embodiment, the magnetic microparticles of this application are carboxyl magnetic microparticles, specifically carboxyl magnetic beads with a particle size of 1 μm. The specific preparation process is as follows: 1 mg of carboxyl magnetic microparticles are mixed with a vortex mixer, placed in an EP tube, 100 mM morpholine ethanesulfonic acid (MES) is added, and after vortex mixing, the tube is placed on a magnetic rack and left to stand for 5 min to separate the carboxyl magnetic microparticles from the liquid and enrich the magnetic microparticles. The supernatant is then removed. 100 mM morpholine ethanesulfonic acid (MES), solid 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), solid N-hydroxysuccinimide (NHS), and solid streptavidin were added sequentially. The mixture was rotated and stirred at 37°C for 2 hours to initiate the coupling reaction. The mass ratio of EDC to NHS was 1:1, and the final concentrations of both EDC and NHS were 1.5 mg / mL. The mass ratio of SA (streptavidin) to carboxylated magnetic microparticles was 1:25, and the final concentration of the carboxylated magnetic microparticles was 5 mg / mL. After the coupling reaction was completed, the EP tube was placed on a magnetic separation rack, the supernatant was removed, blocking solution was added, and the mixture was rotated and stirred at 37°C for 1 hour to initiate the blocking reaction. After sealing, the magnetic microparticles were washed three times with 100 mM MES to obtain streptavidin-modified magnetic microparticles. Finally, the streptavidin-modified magnetic microparticles were stored in a preservation solution with a concentration of 0.3 mg / mL.

[0057] Optionally, the biotin-labeled cytotoxin-associated protein A antibody of this application is prepared by the following steps:

[0058] Take the cytotoxin-associated protein A antibody, add it to PBS buffer, mix well, then add the activated biotin, and rotate the mixture at room temperature in the dark.

[0059] After the reaction was complete, the sample was transferred to a cellulose dialysis bag and dialyzed with PBS buffer to obtain the biotin-labeled cytotoxin-associated protein A antibody.

[0060] In this embodiment, the specific preparation process is as follows: Take 1 mg of cytotoxin-associated protein A (CagA) antibody, add 0.01 M PBS buffer to a total volume of 1 mL to obtain an antibody solution. After mixing, the concentration of the cytotoxin-associated protein A antibody in the antibody solution is 1 mg / mL. Take out the activated biotin, add a certain amount of purified water to the tube according to the mass, so that the final concentration of biotin is 10 mg / mL. Add 20 μL of activated biotin to the antibody solution, mix well, and rotate at room temperature in the dark for 2 hours. After the reaction is completed, transfer the solution to a cellulose dialysis bag, dialyze with 1 L of 0.01 M PBS at 2℃~8℃ for 2 hours to obtain biotin-labeled cytotoxin-associated protein A antibody. Transfer the biotin-labeled cytotoxin-associated protein A antibody to a clean centrifuge tube, measure the volume, add glycerol to a final volume of 2 mL to obtain a concentrated solution of biotin-labeled cytotoxin-associated protein A antibody with a concentration of 0.5 mg / mL.

[0061] In addition, the chemiluminescent material-labeled cytotoxin-associated protein A antibody of this application is prepared by the following steps:

[0062] Take a chemiluminescent substance, add 2-iminothione hydrochloride solution to it, mix well and react at room temperature, add glycine solution to react, desalt and purify to obtain the activated chemiluminescent substance;

[0063] Take the cytotoxin-associated protein A antibody, add a solution of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester sodium salt, vortex mix and react at room temperature, add glycine solution and react, desalt and purify to obtain the activated antibody.

[0064] The activated antibody and the activated chemiluminescent substance were vortexed and mixed, reacted at room temperature, N-ethylmaleimide was added, vortexed and mixed, reacted at room temperature, ethanolamine was added, vortexed and mixed, reacted at room temperature, and then desalted and purified to obtain the purified chemiluminescent substance-labeled cytotoxic protein A antibody.

[0065] In this embodiment, the chemiluminescent substance is alkaline phosphatase, and the specific preparation process is as follows: 100 μg of alkaline phosphatase was added to 5 μL of 2 mg / mL 2-iminothione hydrochloride solution, mixed, and reacted at room temperature for 1 hour. Then, 1 μL of 1 M glycine solution was added and reacted for 10 minutes. The mixture was then desalted and purified using a desalting column to obtain activated alkaline phosphatase. 100 μg of cytotoxic protein A antibody was added to 5 μL of 2 mg / mL 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonyl succinimide ester sodium salt (i.e., Sulfo-SMCC) solution, vortexed, and reacted at room temperature for 1 hour. Then, 1 μL of glycine solution was added and reacted for 10 minutes. The mixture was then desalted and purified using a desalting column to obtain activated antibody. The activated antibody was vortexed with the modified alkaline phosphatase and reacted at room temperature for 1 hour. Then, 10 μL of 10 mg / mL N-ethylmaleimide was added, and the mixture was vortexed and reacted at room temperature for half an hour. Finally, 10 μL of 10 mg / mL ethanolamine was added, and the mixture was vortexed and reacted at room temperature for half an hour. The purified enzyme-labeled product (i.e., alkaline phosphatase-labeled cytotoxic protein A antibody) was obtained by desalting and purifying with a desalting column. 100 μL of 0.01 M PBS was added, and an equal volume of glycerol was added. The mixture was then stored at -20°C.

[0066] Optionally, the blocking solution of this application includes 3 mol / L ethanolamine, 1% bovine serum albumin (BSA), 0.5% Tween-20, the pH of the blocking solution is 8.0, and / or the preservation solution includes PBS buffer containing 0.05% procin-300.

[0067] This application also provides a method for determining cytotoxin-associated protein A, using the above-described kit. Figure 1 As shown, Figure 1 This is a flowchart illustrating one embodiment of the method for determining cytotoxin-associated protein A according to this application. Specifically, it includes the following steps:

[0068] S1: Plot the master calibration curve;

[0069] S2: Test the calibrator, obtain the test results of the calibrator, adjust the master calibration curve according to the test results, and obtain the working calibration curve;

[0070] S3: After confirming the calibration is qualified according to the quality control sample and the working calibration curve, react the sample to be tested with the Ra reagent, Rb reagent, Rc reagent and luminescent substrate, and measure the luminescence value;

[0071] S4: Calculate the concentration of cytotoxic protein A in the sample to be tested based on the luminescence value.

[0072] In this embodiment, this application uses magnetic microparticle chemiluminescence immunoassay to determine cytotoxic protein A in human serum, plasma, or whole blood. Calibration tests are performed using a matching kit and calibrators. Obtaining the master calibration curve involves directly generating it from the master curve card within the kit and scanning and importing the calibrator assignment information into the system. The calibrators are used to detect a series of gradient concentration calibrators using a fully automated chemiluminescence immunoassay analyzer when detecting cytotoxic protein A. The fully automated chemiluminescence immunoassay analyzer adjusts the master calibration curve using a two-point calibration method based on the calibrator test results to obtain the current working calibration curve. Calibration is considered successful if the quality control sample test results fall within the target range of the working calibration curve. This application employs a double-antibody sandwich method, which can be used in a one-step process. The test sample, the reagent bottle containing the Ra reagent, the reagent bottle containing the Rb reagent, the reagent bottle containing the Rc reagent, and the reagent bottle containing the luminescent substrate are placed into a fully automated chemiluminescence immunoassay analyzer. The chemiluminescence immunoassay analyzer performs the mixing reaction, and then the fully automated chemiluminescence immunoassay analyzer measures the luminescence value of the test sample after the reaction. The concentration of cytotoxic protein A in the test sample is calculated based on the relative luminescence units (RLU) of the test sample after the reaction.

[0073] During the detection process, the cleaning solution described in this application is also placed in a designated location within the fully automated chemiluminescence immunoassay analyzer. After the antigen-antibody reaction is complete (i.e., after the reaction of the test sample, Ra reagent, Rb reagent, and Rc reagent), the cleaning solution needs to be added to remove any substances that have not bound to the magnetic microparticle-immune complex. The cleaning effect of the cleaning solution directly affects the accuracy of the detection results. The cleaning solution described in this application contains preservatives, surfactants, and buffer solutions, which can improve the accuracy of the detection results.

[0074] The beneficial effects of this application are:

[0075] The kit described in this application uses a fully automated chemiluminescence immunoassay analyzer as the detection tool, employing chemiluminescence to detect cytotoxin-associated protein A. It offers rapid detection, simple operation, and good reproducibility. The kit utilizes a binding system of streptavidin magnetic microparticles and biotin-labeled derivatives to reduce non-specific adsorption and improve sample accuracy. Simultaneously, it avoids magnetic microparticle aggregation caused by direct antibody coating, thus resolving stability issues. Compared with existing technologies, the kit presented in this application offers advantages such as rapid testing, good reproducibility, stable reagent performance, high sensitivity, and accurate results.

[0076] This application also provides performance evaluation of the kit based on the determination of cytotoxic-associated protein A.

[0077] Sensitivity testing: Following the experimental protocol recommended in CLSI EP17-A, the sensitivity of the kit for measuring cytotoxic protein A was calculated to be 1.5 pg / mL.

[0078] Linearity detection: Linearity analysis was performed on samples with concentrations of 3.00 pg / mL, 10.00 pg / mL, 100.00 pg / mL, 1000.00 pg / mL, and 4000.00 pg / mL. The linear correlation coefficients are shown in Table 1. In addition, the linear range of this kit is 3 to 4000 pg / mL.

[0079] Table 1 Linearity Detection

[0080]

[0081] Precision determination: Low concentration samples of cytotoxic protein A (CPA) at approximately 10.00 pg / mL and high concentration samples of CPA at approximately 1000.00 pg / mL were taken, and 10 parallel tests were performed for each sample and each concentration. Three batches of kits were used for detection, and the intra-batch and inter-batch differences of the kits were calculated as shown in Table 2. The results showed that the intra-batch and inter-batch differences of the kits were both <10%.

[0082] Table 2 Precision Testing

[0083]

[0084]

[0085] Interference assay: Mixed serum samples were added with interfering agents, including bilirubin, hemoglobin, triglycerides, and rheumatoid factor. The concentrations were bilirubin (30 mg / dL), hemoglobin (350 mg / dL), triglycerides (500 mg / dL), and rheumatoid factor (450 I U / mL). Serum samples with and without interfering agents were measured, and the test deviations were calculated (see Table 3). The deviations were <±15%. The results indicate that the interference levels meet the NCCLS documentation standards and can be used for accurate assessment of cytotoxic protein A status in clinical laboratories.

[0086] Table 3 Interference Experiments

[0087]

[0088] Sensitivity comparison experiment: Zero-concentration calibrators or sample dilutions were detected using magnetic microparticle chemiluminescence immunoassay. The measurements were repeated 20 times to obtain the RLU values. The average value and relative deviation were calculated to obtain the M-2SD. These values ​​were then substituted into the calibration curve to obtain the corresponding concentration values, as shown in Table 4. The concentration value obtained using magnetic microparticle chemiluminescence immunoassay was 1.5 pg / mL, which is approximately 6 times higher than the detection limit of 10 pg / mL of the traditional enzyme-linked immunosorbent assay (ELISA).

[0089] Table 4 Sensitivity Experiment

[0090]

[0091]

[0092] It should be understood that although the steps in the flowcharts of the accompanying figures are shown sequentially as indicated by the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless explicitly stated herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least some steps in the flowcharts of the accompanying figures may include multiple sub-steps or multiple stages. These sub-steps or stages 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 in turn with other steps or at least some of the sub-steps or stages of other steps.

[0093] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A kit for determining cytotoxin-associated protein A, characterized in that, For the detection of cytotoxic protein A in human serum, plasma or whole blood, including: Ra reagent, Rb reagent, Rc reagent and luminescent substrate; The Ra reagent includes streptavidin-modified magnetic microparticles; The Rb reagent includes a biotin-labeled antibody against cytotoxic protein A; The Rc reagent includes a chemiluminescent antibody labeled with cytotoxic-associated protein A. The kit also includes cleaning solution, calibrators, and quality control materials; The cleaning solution consists of 15.76 mg / mL Tris-HCl, 0.5 mg / mL Proclin 300, and 0.001 mg / mL Tween-20. The concentration of the streptavidin-modified magnetic microparticles is ≥0.3 mg / mL; The concentration of the biotin-labeled cytotoxic protein A antibody is ≥10 μg / mL; The concentration of the chemiluminescent material-labeled cytotoxic protein A antibody is ≥5 μg / mL; The luminescent substrate comprises 0.1 mg / mL of a 1,2-dioxane derivative; The biotin includes N-hydroxysuccinimide biotin; The chemiluminescent material includes alkaline phosphatase; The streptavidin-modified magnetic microparticles have a particle size of 1~3 μm.

2. The kit for determining cytotoxin-associated protein A according to claim 1, characterized in that, The streptavidin-modified magnetic microparticles are prepared by the following steps: After mixing the magnetic microparticles with a vortex mixer, morpholine ethanesulfonic acid was added to enrich the magnetic microparticles, and the supernatant was removed. Morpholin ethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and streptavidin were added sequentially, and the mixture was rotated to mix and react. After the reaction is complete, place the mixture on a magnetic rack, discard the supernatant, add the blocking liquid, and rotate to mix the reaction to obtain the streptavidin-modified magnetic microparticles.

3. The kit for determining cytotoxin-associated protein A according to claim 1, characterized in that, The biotin-labeled cytotoxin-associated protein A antibody was prepared via the following steps: Add the cytotoxin-associated protein A antibody to PBS buffer, mix well, then add activated biotin and rotate at room temperature in the dark. After the reaction was complete, the sample was transferred to a cellulose dialysis bag and dialyzed with PBS buffer to obtain the biotin-labeled cytotoxin-associated protein A antibody.

4. The kit for determining cytotoxin-associated protein A according to claim 1, characterized in that, The chemiluminescent material-labeled cytotoxin-associated protein A antibody was prepared via the following steps: Take a chemiluminescent material, add 2-iminothionane hydrochloride solution to it, mix well and react at room temperature, then add glycine solution to react and obtain an activated chemiluminescent material. Take the cytotoxin-associated protein A antibody, add a solution of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester sodium salt, vortex mix and react at room temperature, add glycine solution and react to obtain the activated antibody. The activated antibody and the activated chemiluminescent substance were vortexed and mixed, reacted at room temperature, N-ethylmaleimide was added, vortexed and mixed, reacted at room temperature, ethanolamine was added, vortexed and mixed, reacted at room temperature, and then desalted and purified to obtain the purified chemiluminescent substance-labeled cytotoxic protein A antibody.

5. A method for determining cytotoxin-associated protein A for non-diagnostic purposes, characterized in that, The assay was performed using the kit as described in any one of claims 1 to 4.

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