A rapid assay for soluble st2 content using a magnetic particle chemiluminescent assay kit

Abstract

The application discloses a magnetic particle chemiluminescence detection kit for rapidly determining soluble ST2 content, which comprises magnetic separation reagent, calibrant, quality control product and luminescence substrate liquid, and is free of reagent R1 and reagent R2. The magnetic separation reagent is a mixed solution containing magnetic particles and acridinium ester, wherein the surface of the magnetic particles is coated with anti-ST2 antibody 1, and the anti-ST2 antibody 2 is labeled with acridinium ester. The kit is designed for the detection of soluble growth stimulating expression gene 2 protein. The magnetic separation reagent is a mixed solution of magnetic particles and acridinium ester, and the reagent component is only one mixed reagent, which is free of reagent R1 and reagent R2, so that the detection time is greatly shortened. Meanwhile, a blocking agent is added into the buffer solution of the magnetic separation reagent, so that the influence of complement and heterophilic antibody is effectively eliminated, and the sample detection accuracy is improved.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic reagents, and in particular to a magnetic microparticle chemiluminescence detection kit that uses magnetic microparticle chemiluminescence technology and antigen-antibody binding technology to detect the content of soluble growth-stimulating gene 2 protein. Background Technology

[0002] Soluble growth hormone-stimulated gene 2 (ST2) protein is a myocardial protein produced by cardiomyocytes under biomechanical stress. Studies have found that ST2 can be expressed by cardiac fibroblasts and cardiomyocytes and is a myocardial protein induced by biomechanical stress. It participates in various pathophysiological processes and has immunomodulatory functions, playing a particularly important role in various inflammatory and allergic diseases. Furthermore, quantitative measurement of ST2 levels helps clinicians better assess the prognostic risk of heart failure patients.

[0003] Published research indicates that patients with ST2 levels above the clinical threshold have a high risk of death, while those with ST2 levels below the threshold have a much lower mortality rate. Therefore, ST2 levels can be used to predict mortality. Current research also shows that ST2 concentration is related to the severity of heart failure; a concentration above 35.0 ng / mL indicates a higher risk of poor prognosis. In patients with acute coronary syndrome, ST2 levels above 35.0 ng / mL have a 3 times higher risk of death or progression to heart failure within 30 days compared to those with ST2 levels below the threshold, and a 2.3 times higher relative risk within one year. Furthermore, current research suggests that ST2 participates in myocardial remodeling and can be used to help assess the effectiveness of anti-fibrotic drugs such as eplerenone.

[0004] Regarding current ST2 detection technologies, Li Denghong et al.'s paper, "Research Progress on Detection Methods of Soluble Growth Stimulating Gene Expression 2 Protein," mentions several methods for ST2 detection: ELISA, chromatography, two-color upconversion nanoparticle fluorescence immunoassay, and chemiluminescence. The first three methods suffer from low sensitivity, narrow linear range, poor repeatability, and low quantitative accuracy. Magnetic microparticle chemiluminescence, on the other hand, is characterized by simple operation, high sensitivity, wide linear range, and good repeatability. However, existing magnetic microparticle chemiluminescence methods, based on the double-antibody sandwich principle, often require kits with two reagent components: a magnetic microparticle reagent component and a catalytic enzyme or acrid ester reagent component. This necessitates two samplings during detection, increasing operation time and complexity. Furthermore, in existing patented ST2 magnetic microparticle chemiluminescence detection kits, the selected magnetic microparticles all possess carboxyl active groups on their surface. During antibody coating, an active agent (EDC) is needed to activate these surface groups. However, EDC is hygroscopic and must be prepared fresh for each use, further complicating the operation. Summary of the Invention

[0005] The present invention aims to develop a magnetic microparticle chemiluminescence detection kit for determining the content of soluble growth-stimulating gene 2 protein, which is simple to prepare, highly sensitive, pollution-free, easy to operate, rapid, specific, and inexpensive.

[0006] Based on the above objectives, and building upon the magnetic microparticle chemiluminescence platform, this invention provides a single-reagent component assay kit for the rapid determination of soluble growth-stimulating gene 2 protein content. This kit eliminates reagents R1 and R2; the single reagent component is a mixture of magnetic microparticles and acridine ester, referred to as the magnetic separation reagent. The magnetic microparticles are coated with anti-ST2 antibody 1, and the acridine ester is labeled with anti-ST2 antibody 2. Furthermore, the kit includes six calibrators containing different concentrations of ST2 antigen, and two quality control samples containing different concentrations of ST2 antigen. The luminescent substrate solution is an oxidized acridine ester substrate solution.

[0007] Acridinium esters are a general term for acridinium salts, referring to a class of chemical substances used as chemiluminescent markers. There are different series of acridinium esters. These substances usually have low background luminescence and can be used to detect samples with low or trace concentrations in the reaction. They are a class of luminescent agents with high luminescence efficiency, characterized by rapid luminescence, high luminescence value, and sensitive detection. Therefore, they are widely recognized in the field of immunoassay and are increasingly being used in immunoassay reagents.

[0008] The magnetic microparticles have toluenesulfonyl groups on their surface, eliminating the need for activators such as EDC during the coating process. Phosphate buffer is used instead of ammonium sulfate buffer, eliminating the need for fresh preparation and simplifying the operation.

[0009] The method for preparing the concentrated solution of anti-ST2 antibody 1 coated on the surface of the magnetic microparticles is as follows:

[0010] (1) Prepare a 1:0.1M sodium borate buffer solution for coupling, with a pH of 9.5, and store at 2-8℃;

[0011] (2) Prepare coupling buffer 2: 2M potassium phosphate buffer, pH 8.5, and store at 2-8℃;

[0012] (3) Preparation of coupling blocking solution: 0.2M phosphate buffer, pH 7.5, containing 1% bovine serum albumin, 0.1% Tween 20, 10% sucrose, and 0.2% Bronidox;

[0013] (4) Preparation of washing and storage solution: 0.2M phosphate buffer, pH 7.5, containing 1% bovine serum albumin and 10% sucrose (by mass);

[0014] (5) Resuspension of toluenesulfonyl-modified magnetic microspheres by vortex oscillation;

[0015] (6) Transfer 100 μL of toluenesulfonyl-modified magnetic beads to a 2 mL centrifuge tube and add 1 mL of coupling buffer 1.

[0016] (7) Place the centrifuge tube on the magnetic separator until the magnetic beads migrate toward the side closer to the magnet, until the solution becomes clear;

[0017] (8) Carefully remove the supernatant to avoid suspending the magnetic beads and microspheres;

[0018] (9) Remove the centrifuge tube from the magnet and resuspend the magnetic beads in 1 mL of coupling buffer 1 by vortexing for 5 minutes.

[0019] (10) Repeat steps (7)-(9) 3 times;

[0020] (11) Use coupling buffer 1 to weigh the magnetic beads to 100 μL, add 322 μL of coupling buffer 2 and 400 μg of ST2 antibody 1, and then use coupling buffer 1 to weigh to 1 mL.

[0021] (12) After thorough shaking and mixing, incubate overnight in an incubator at 37°C to obtain the coupling reaction system;

[0022] (13) Add 5 μL of ethanolamine to the coupling reaction system after incubation overnight in step (12) in a fume hood, vortex shake, and then mix on a vortex mixer for 30 minutes to terminate the reaction.

[0023] (14) Place the centrifuge tube on the magnetic separator until the microspheres migrate toward the side closer to the magnet, until the solution becomes clear;

[0024] (15) Carefully remove the supernatant to avoid suspending the magnetic beads and microspheres;

[0025] (16) Remove the centrifuge tube from the magnet and resuspend the magnetic beads in 1 mL of coupling buffer 1 by vortexing for 5 minutes.

[0026] (17) Repeat steps (14)-(16) 3 times;

[0027] (18) Add 1 mL of coupling blocking solution, vortex for 5 minutes to resuspend the magnetic beads, and incubate overnight in a 37°C incubator.

[0028] (19) Place the centrifuge tube on the magnetic separator until the microspheres migrate toward the side closer to the magnet, until the solution becomes clear;

[0029] (20) Carefully remove the supernatant to avoid suspending the magnetic beads and microspheres;

[0030] (21) Remove the centrifuge tube from the magnet and resuspend the magnetic beads in 1 mL of washing and storage solution by vortexing for 5 minutes.

[0031] (22) Repeat steps (19)-(21) 3 times. The coupled magnetic beads are weighed to 1 mL with washing and storage solution to obtain magnetic microparticle concentrate. Store it for later use to obtain magnetic microparticle concentrate with a concentration of 10 mg / mL, which is the concentrate of anti-ST2 antibody 1 coated on the surface of magnetic microparticles.

[0032] (23) The concentration of the magnetic microparticle concentrate prepared above is 10 mg / mL, and the technical data of the reaction system is 1 mL. The corresponding parameters and the amount of coupling buffer added are increased proportionally with the reaction system.

[0033] The acridinium ester-labeled soluble growth-stimulating gene 2 protein monoclonal antibody technology is a commonly used conjugation process in chemiluminescence platforms. Acridinium ester-labeled anti-ST2 antibody 2 was obtained according to existing technology.

[0034] Preparation of preferred magnetic separation reagent: Dilute the 10 mg / mL magnetic microparticle concentrate to 1 mg / mL with magnetic separation reagent buffer and equilibrate at 2-8℃ for 24 h; add 5 μg of acridine ester-labeled anti-ST2 antibody 2 (0.5 μg / mL acridine ester-labeled anti-ST2 antibody 2, 10 mL reaction system, total 5 μg), and mix at room temperature for 0.5 h.

[0035] Preferably, the magnetic separation reagent buffer solution has a pH of 5.8–8.5 and includes Tris (5.98–6.52 g / L); Proclin-300 (0.98–1.99 g / L); Thimerosal (0.99–2.01 g / L); Sodium chloride (8.5–9.5 g / L); Bovine serum albumin (10–30 g / L); Gelatin (5–10 g / L); N-ethylcis-butenedimide (0.85–2.05 g / L); Triton X-100 (0.98–1.99 g / L); Tween-20 (0.98–1.99 g / L); and the remainder is purified water.

[0036] Preferably, the magnetic separation reagent buffer has a pH of 7.4 and includes Tris (6.05 g / L), Proclin-300 (1.99 g / L), thimerosal (0.99 g / L), sodium chloride (8.98 g / L), bovine serum albumin (29.88 g / L), gelatin (9.86 g / L), N-ethylcis-butenedimide (1.05 g / L), Triton X-100 (0.98 g / L), Tween-20 (1.99 g / L), and the remainder is purified water.

[0037] The magnetic separation reagent contains an inhibitor, preferably HBR-2-Purfied at a concentration of 40–60 μg / mL; HBR-20-Purfied at a concentration of 40–60 μg / mL; or HBR Plus at a concentration of 10–30 μg / mL.

[0038] More preferably, the magnetic separation reagent contains HBR-2-Pμrfied at a concentration of 50 μg / mL; HBR-20-Pμrfied at a concentration of 50 μg / mL; and HBR Plμs at a concentration of 20 μg / mL.

[0039] Traditional reagent kits contain at least two reagent components: antibody 1 coated on the surface of magnetic microparticles and antibody 2 labeled on acrid esters. These components are stored separately and require separate sampling during use. In this application, the magnetic separation reagent is composed of a direct mixture of the two reagent components, requiring only one sampling during use, saving time and increasing sample throughput.

[0040] Preferably, the calibration solution series consists of diluents containing different concentrations of ST2 antigen.

[0041] Preferably, the buffer solution in the calibrator series has a pH of 7.0–8.0, and the buffer components include PBS at a concentration of 0.02M; Casein at a concentration of 10–30 g / L; N-ethylcis-butenedimide at a concentration of 0.85–2.05 g / L; Proclin-300 at a concentration of 1.98–1.99 g / L; tetracycline at a concentration of 0.98–0.99 g / L; and the remaining component is purified water.

[0042] Preferably, the calibrator solution series has a concentration range of 0–300 ng / mL, a buffer pH of 7.4, and the buffer components include PBS at a concentration of 0.02 M; Casein at a concentration of 20 g / L; N-ethylcis-butenedimide at a concentration of 1.05 g / L; Proclin-300 at a concentration of 1.98 g / L; tetracycline at a concentration of 0.99 g / L; and the remaining component is purified water.

[0043] Preferably, the different concentrations of the calibrator solution series are 0 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 150 ng / mL, and 300 ng / mL.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] This invention's kit is designed for the detection of soluble growth-stimulating gene 2 protein. It is a magnetic microparticle chemiluminescence detection kit. The magnetic separation reagent is a mixture of magnetic microparticles and acridine ester. The reagent components are only a single mixture, eliminating reagents R1 and R2, significantly reducing detection time. Simultaneously, blocking agents (HBR-2-Purfied, HBR-20-Purfied, HBR Plus) are added to the buffer of the magnetic separation reagent, effectively eliminating the influence of complement, heterophile antibodies, etc., and improving sample detection accuracy. Furthermore, this invention uses magnetic microparticles with toluenesulfonyl groups on their surface, eliminating the need for activators such as EDC and avoiding the need for on-site preparation. Compared with the magnetic microparticle coating technology disclosed in existing ST2 kit patents, the coating process is simpler and more conducive to process stability.

[0046] The reagents selected in this invention have been experimentally found to have no non-specific cross-linking between the two antibodies in the magnetic separation reagent, which can ensure the accuracy of the detection results. At the same time, the selected buffer system and the various substances added are suitable for magnetic microparticles, acrid esters and the two antibodies, without affecting the stability of the entire system.

[0047] The magnetic microparticle chemiluminescence assay kit for detecting the content of soluble growth-stimulating gene 2 protein provided by this invention can be used in conjunction with a fully automated chemiluminescence analyzer, greatly simplifying the operation steps, increasing detection speed and throughput, improving detection efficiency, and avoiding errors caused by human operation. This kit exhibits good specificity and repeatability, uses a less expensive acridil ester chemiluminescence system instead of an alkaline phosphatase chemiluminescence system, resulting in lower costs, high detection accuracy (sensitivity reaching 0.0055 ng / mL), short reaction time (reduced from 30 minutes to 15 minutes), and good stability, making it well-suited for ST2 detection and better meeting the comprehensive performance requirements of the market. The results of the examples show that the blank limit of this kit is 0.0055 ng / mL, the intraanalytical precision is 1.34%, and the interanalytical precision is 1.16%, demonstrating good precision. It exhibits strong anti-interference ability, remaining unaffected by the determination of 5 g / L hemoglobin, 0.5 mmol / L total bilirubin, and 10.0 g / L fat emulsion. It shows good correlation with commercially available products, and the reagent exhibits good stability.

[0048] This application uses only one reagent component (excluding calibrators, quality control materials, and substrates). The reaction mode has been changed from the original two-step method to a one-step method, reducing the sample addition time and incubation time. At the same time, the acridinium ester luminescence system is a flash type, reducing the counting time from the original 90 seconds to 1-2 seconds, resulting in high detection efficiency. Attached Figure Description

[0049] Figure 1 This is a graph showing the concentration-luminescence value of soluble growth-stimulating gene 2 protein in the kit of Example 1 of the present invention.

[0050] Figure 2 This is a graph showing the correlation between the reagent kit of Example 1 and the reagent kit of Comparative Example 2 in serum sample detection.

[0051] Figure 3 This is a graph showing the correlation between the reagent kit of Example 1 and the reagent kit of Comparative Example 2 in plasma sample detection.

[0052] Figure 4 This is a graph showing the correlation between the serum sample detection results of the kit in Comparative Example 1 and the kit in Comparative Example 2 of the present invention.

[0053] Figure 5 This is a graph showing the correlation between the reagent kit of Comparative Example 1 and the reagent kit of Comparative Example 2 in plasma sample detection. Detailed Implementation

[0054] The present invention will be further explained below with reference to the embodiments and accompanying drawings, but this is not intended to limit the scope of protection of this application.

[0055] The magnetic microparticle chemiluminescence detection kit for determining the content of soluble growth-stimulating gene 2 protein of the present invention comprises magnetic separation reagent, calibrator, quality control material and chemiluminescent substrate solution.

[0056] The magnetic separation reagent includes: 1) two antibodies: ST2 monoclonal antibody 1 coated on the surface of immunomagnetic beads and acridine ester-labeled anti-ST2 antibody 2, with a magnetic particle concentration of 1 mg / mL and acridine ester-labeled anti-ST2 antibody 2 concentration of 0.5 μg / mL;

[0057] 2) Buffer solution: The magnetic separation reagent buffer solution has a pH of 7.4 and includes Tris (6.05 g / L), Proclin-300 (1.99 g / L), thimerosal (0.99 g / L), sodium chloride (8.98 g / L), bovine serum albumin (29.88 g / L), gelatin (9.86 g / L), N-ethylcis-butenedimide (1.05 g / L), Triton X-100 (0.98 g / L), Tween-20 (1.99 g / L), and purified water as the remainder.

[0058] The calibrators include: 1) a dilution of soluble growth-stimulating gene 2 protein antigen; 2) a buffer solution: the buffer solution consists of PBS (0.02 M); Casein (10–30 g / L); N-ethylcis-butenedimide (0.85–2.05 g / L); Proclin-300 (1.98–1.99 g / L); tetracycline (0.98–0.99 g / L); and purified water as the remaining component. The pH of the calibrator buffer solution is 7.4. The calibrators are a series of reagents containing different concentrations (0, 5, 20, 50, 150, 300 ng / mL).

[0059] A method for preparing a rapid magnetic particle chemiluminescence detection kit for determining the content of soluble ST2, for non-disease diagnostic purposes, includes the following steps:

[0060] Preparation of magnetic separation reagent: 1) Prepare coupling buffer, blocking solution and storage solution, wash and activate the magnetic beads, then wash, couple and block the supernatant of magnetic separation to obtain immunomagnetic beads coated with ST2 monoclonal antibody 1; 2) Adjust the volume of the immunomagnetic beads coated with ST2 monoclonal antibody 1 with the storage solution of magnetic separation reagent to obtain magnetic microparticle concentrate; dilute the magnetic microparticle concentrate to a specific concentration using magnetic separation reagent buffer; 3) Add 5 μg of acridinium ester-labeled anti-ST2 antibody 2 (10 mL reaction system) and mix at room temperature for 0.5 hours.

[0061] Preparation of calibrators: 1) Prepare phosphate buffer according to the component content of the calibrator buffer; 2) Dissolve ST2 antigen in the calibrator buffer to prepare different concentrations.

[0062] Determination method and standard curve plotting of ST2 content using a magnetic particle chemiluminescence detection kit:

[0063] (1) First, add 20 μL of the calibrator series (concentrations of 0, 5, 20, 50, 150, 300 ng / mL) and 50 μL of magnetic separation reagent to the reaction tube in sequence, and mix and incubate at 37°C for 10 min.

[0064] (2) Wash three times with washing solution to remove unbound antibodies and impurities;

[0065] (3) Add 100 μL of each luminescent substrate excitation solution. After the acridinium ester catalyzes the luminescence of the substrate, the relative luminescence intensity (RLU) is measured using a chemiluminescence detector from Leadman Corporation to obtain a series of values ​​for ST2 concentration and luminescence value.

[0066] (6) The standard curve of soluble growth-stimulating gene 2 protein concentration-luminescence value was obtained by fitting the series of values ​​of soluble growth-stimulating gene 2 protein concentration-luminescence value.

[0067] (7) Calculate and determine the concentration value of the sample to be tested based on the fitted standard curve of soluble growth-stimulating gene 2 protein concentration-luminescence value.

[0068] Within a certain range, RLU is directly proportional to the concentration of soluble growth-stimulating gene 2 protein antigen. The content of soluble growth-stimulating gene 2 protein in the test sample can be read from the standard curve by interpolation.

[0069] Example 1: A rapid determination kit for chemiluminescence detection of soluble ST2 content using magnetic microparticles includes magnetic separation reagents, calibrators, and chemiluminescent substrate solution.

[0070] In this embodiment, the magnetic separation reagent includes: 1) magnetic microparticles: immunomagnetic beads coated with soluble monoclonal antibody against growth-stimulating gene 2 protein (anti-ST2 antibody 1), with a concentration of 1 mg / mL; 2) acridinium-labeled anti-ST2 antibody 2: acridinium-labeled soluble monoclonal antibody against growth-stimulating gene 2 protein, with a concentration of 0.5 μg / mL; 3) stock solution: 0.2 M phosphate buffer, pH 7.5, containing 1% bovine serum albumin and 10% sucrose; 4) magnetic separation reagent buffer.

[0071] In this embodiment, the calibrators include: 1) dilutions containing different concentrations of soluble growth-stimulating gene 2 protein antigen; 2) buffer solutions: PBS, 0.02M; Casein, 20 g / L; N-ethylcis-butenedimide, 1.05 g / L; Proclin-300, 1.98 g / L; tetracycline, 0.99 g / L; and purified water as the remaining component. The pH of the buffer solution in the calibrator series is 7.4. The calibrator series consists of reagents containing different concentrations (0, 5, 20, 50, 150, 300 ng / mL).

[0072] Example 2: Preparation and determination method of a magnetic microparticle chemiluminescence detection kit for rapid determination of soluble ST2 content:

[0073] (1) First, 20 μL of the calibrator series of Example 1 (concentrations of 0, 5, 20, 50, 150, 300 ng / mL) and 50 μL of magnetic separation reagent of Example 1 were added to the reaction tubes respectively, and mixed and incubated at 37°C for 10 min.

[0074] (2) Wash three times with washing solution to remove unbound antibodies and impurities;

[0075] (3) Add 100 μL of the luminescent substrate excitation solution from Example 1. After the acrid ester catalyzes the luminescence of the substrate, the relative luminescence intensity (RLU) is measured using a Leadman chemiluminescence detector. The results are shown in the table below:

[0076] The relative luminescence intensity results of each calibrator are shown in Table 1.

[0077] Table 1

[0078] Calibrator (ng / mL) 0 5 20 50 150 300 RLU(100000) 0.02 3.23 12.92 32.35 96.92 193.81

[0079] (4) Fit the values ​​in Table 1 to obtain ( Figure 1 The standard curve of soluble growth-stimulating gene 2 protein concentration versus luminescence value is shown in the figure. The vertical axis represents the luminescence value, and the horizontal axis represents the soluble growth-stimulating gene 2 protein concentration.

[0080] (5) Add 15 μL of the sample to be tested and 60 μL of the magnetic separation reagent mixture from Example 1 into the reaction tube in sequence, and incubate at 37°C for 10 min.

[0081] (6) Wash three times with washing solution to remove unbound antibodies and impurities;

[0082] (7) Add 200 μL of acridine ester luminescent substrate excitation solution to each of the acridine esters. After the acridine esters catalyze the luminescence of the substrates, the relative luminescence intensity is measured using a Leadman self-developed chemiluminescence detector.

[0083] (8) Calculate the soluble growth stimulation expressed gene 2 protein concentration value corresponding to the luminescence value of the test sample according to the standard curve of soluble growth stimulation expressed gene 2 protein concentration - luminescence value in step (3).

[0084] When using the above detection kit of the present invention to determine the content of soluble growth stimulation expressed gene 2 protein, the methodological identification data can reach the following indicators:

[0085] The blank limit is ≤0.0055 ng / mL;

[0086] The linear detection range of soluble growth stimulation expressed gene 2 protein is 1.0 - 300 ng / mL;

[0087] Precision - The average within-run precision is 1.34% (n = 10), and the average between-run precision is 1.16% (n = 10). The precision is much lower than the national requirements, indicating that the kit of the present invention has good repeatability during the experiment;

[0088] Accuracy - The recovery rate is 90% - 100% when the high-value serum with known soluble growth stimulation expressed gene 2 protein concentration is diluted with the low-value serum with known soluble growth stimulation expressed gene 2 protein concentration at a ratio of 1:9;

[0089] Endogenous interference - Hemoglobin < 5 g / L; total bilirubin < 0.5 mmol / L; lipid emulsion < 10.0 g / L; the determination is not interfered, and the deviation of the detection results is less than 10%;

[0090] Specificity - There is no significant cross-reaction with 30 ng / mL of cardiac troponin I (cTnI) and 30 ng / mL of N-terminal pro-brain natriuretic peptide (NT-proBNP);

[0091] There is no high-dose hook effect when the HOOK-ST2 concentration is as high as 100000 ng / mL.

[0092] Comparison: Comparison of the minimum detection limits of different markers and reaction processes

[0093] Using the kits of Example 1 and Comparative Examples 1 - 2, the concentration - RLUs results between the zero-concentration calibrator and the adjacent-concentration calibrator are detected, and a linear equation is obtained by two-point regression fitting to investigate the minimum detection concentration of different kits. Among them, Comparative Example 1 is the Leadman ST2 kit (registration number: Beijing Medical Device Registration 20192400342), and Comparative Example 2 is the ST2 enzyme immunoassay kit of CriticalDiagnostics (registration number: National Medical Device Import Registration 20152400566). The detection results of the 3 kits are shown in Table 2.

[0094] Table 2

[0095] Reagent test kit Limit of detection (ng / mL) Example 1 0.0055 Comparative Example 1 0.158 Comparative Example 2 1.8

[0096] The minimum detection limit data for Example 1 are shown in Table 3 below.

[0097] Table 3

[0098]

[0099] The minimum detection limit data for Comparative Example 1 are shown in Table 4 below.

[0100] Table 4

[0101]

[0102] Comparison 2: Comparison of samples within the range of 0–100 ng / mL tested with different reaction markers and reaction procedures

[0103] Using the kits of Example 1 and Comparative Examples 1-2, samples in the range of 0-100 ng / mL were detected simultaneously. The concentrations of ST2 in human serum / plasma determined by the kits of Example 1 / Comparative Example 1 were plotted on the ordinate, and the results determined by the kit of Comparative Example 2 were plotted on the abscissa. The detection results of the three kits are shown in Table 5.

[0104] Table 5

[0105]

[0106] The measurement results of the samples from Example 1, Comparative Example 1, and Comparative Example 2 are shown in Table 6 below.

[0107] Table 6

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] The results of the conformity rate of the measured values ​​of the samples in Example 1, Comparative Example 1 and Comparative Example 2 are shown in Table 7 below.

[0114] Table 7

[0115]

[0116] The correlation results of serum / plasma sample measurements in Example 1, Comparative Example 1, and Comparative Example 2 are as follows: Figure 2 , 3 4, 5.

[0117] The kit in Example 1 showed good consistency with the detection results of Comparative Example 2, with a correlation R of R between serum values ​​in the range of 0–100 ng / mL. 2 =0.9871( Figure 2 ), plasma measurement value R 2 =0.9868 ( Figure 3 );

[0118] The correlation R between the reagent kit of Comparative Example 1 and the serum measurements of Comparative Example 2 in the range of 0–100 ng / mL 2 =0.8777( Figure 4 ), plasma measurement value R 2 =0.8649 ( Figure 5 The correlation was slightly worse than that of Example 1.

[0119] Table 8 shows a comparison of the reaction time and process of Example 1, Comparative Example 1, and Comparative Example 2.

[0120] Table 8

[0121] Reagent test kit reaction time Adding samples Example 1 15 minutes Step 1 Comparative Example 1 30 minutes 3 steps Comparative Example 2 >4 hours 5 steps

[0122] The reagent kit of Example 1 has few reagent components, a simple sample addition process, and a short reaction time.

[0123] Using the kit from Example 1, the kit's real-time stability was assessed within its shelf life. Performance evaluations were performed at 0, 1, 3, 6, 9, 12, and 13 months, including the limit of detection and linear range. The results are shown in Tables 9 and 10.

[0124] Table 9

[0125] time Limit of detection (ng / mL) 0 months 0.0055 3 months 0.0052 6 months 0.0053 9 months 0.005 12 months 0.0052 13 months 0.0055

[0126] Table 10

[0127]

[0128] Table 9 shows that the limit of detection does not exceed approximately 0.0055 ng / mL within 13 months. Table 10 shows that the linear range remains stable within 13 months. This indicates that the components in the magnetic bead separation reagent of this invention have stable properties and do not change over time.

[0129] The above steps demonstrate that the double-antibody sandwich reaction mode adopted in this invention utilizes the principle of combining chemiluminescence detection technology with magnetic microparticle immunoassay to quantitatively detect the content of soluble growth-stimulating gene 2 (ST2) protein in human serum or plasma samples. The reagent components consist only of magnetic separation reagent, ensuring high detection sensitivity, no pollution, high specificity, speed, simple operation, low requirements for sample pretreatment, and rapid high-throughput detection of large batches of samples, making it convenient for clinical reagent applications.

[0130] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples. Within the framework of this invention, technical features of the above embodiments or different embodiments can also be combined, and many other variations of the different aspects of the invention as described above exist, which are not provided in the details for the sake of brevity. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0131] Any aspects not covered in this invention are applicable to existing technologies.

Claims

1. A rapid magnetic microparticle chemiluminescence detection kit for determining the content of soluble ST2, comprising magnetic separation reagent, calibrator, quality control sample, and luminescent substrate solution, characterized in that, The kit does not contain reagents R1 and R2. The magnetic separation reagent is a mixture containing magnetic microparticles and acridine ester, wherein the surface of the magnetic microparticles is coated with anti-ST2 antibody 1, and the acridine ester is labeled with anti-ST2 antibody 2. The method for preparing the concentrated solution of anti-ST2 antibody 1 coated on the surface of the magnetic microparticles is as follows: (1) Prepare a 1:0.1M sodium borate buffer solution for coupling, with a pH of 9.5, and store at 2-8℃; (2) Prepare coupling buffer 2: 2M potassium phosphate buffer, pH 8.5, and store at 2-8℃; (3) Preparation of coupling blocking solution: 0.2M phosphate buffer, pH 7.5, containing 1% bovine serum albumin, 0.1% Tween 20, 10% sucrose, and 0.2% Bronidox; (4) Preparation of washing and storage solution: 0.2M phosphate buffer, pH 7.5, containing 1% bovine serum albumin and 10% sucrose; (5) Resuspension of toluenesulfonyl-modified magnetic beads by vortex oscillation; (6) Transfer 100 μL of toluenesulfonyl-modified magnetic beads to a 2 mL centrifuge tube and add 1 mL of coupling buffer 1. (7) Place the centrifuge tube on the magnetic separator until the magnetic beads migrate toward the side closer to the magnet, until the solution becomes clear; (8) Carefully remove the supernatant to avoid suspending the magnetic beads; (9) Remove the centrifuge tube from the magnet and resuspend the magnetic beads in 1 mL of coupling buffer 1 by vortexing for 5 minutes; (10) Repeat steps (7)-(9) 3 times; (11) Use coupling buffer 1 to weigh the magnetic beads to 100 μL, add 322 μL of coupling buffer 2 and 400 μg of ST2 antibody 1, and then use coupling buffer 1 to weigh to 1 mL. (12) After thorough shaking and mixing, incubate overnight in a 37°C incubator to obtain the coupling reaction system; (13) Add 5 μL of ethanolamine to the coupling reaction system after incubation overnight in step (12) in a fume hood, vortex shake, and then mix on a vortex mixer for 30 minutes to terminate the reaction. (14) Place the centrifuge tube on the magnetic separator until the microspheres migrate toward the side closer to the magnet, until the solution becomes clear; (15) Carefully remove the supernatant to avoid suspending the magnetic beads; (16) Remove the centrifuge tube from the magnet and resuspend the magnetic beads in 1 mL of coupling buffer 1 by vortexing for 5 minutes; (17) Repeat steps (14)-(16) 3 times; (18) Add 1 mL of coupling blocking solution, vortex for 5 minutes to resuspend the magnetic beads, and incubate overnight in a 37°C incubator. (19) Place the centrifuge tube on the magnetic separator until the microspheres migrate toward the side closer to the magnet, and until the solution becomes clear; (20) Carefully remove the supernatant to avoid suspending the magnetic beads; (21) Remove the centrifuge tube from the magnet and resuspend the magnetic microspheres in 1 mL of washing and storage solution by vortexing for 5 minutes; (22) Repeat steps (19)-(21) 3 times, and weigh the coupled magnetic beads to 1 mL with washing and storage solution to obtain magnetic microparticle concentrate. Store it for later use to obtain magnetic microparticle concentrate with a concentration of 10 mg / mL, which is the concentrate of anti-ST2 antibody 1 coated on the surface of magnetic microparticles. Preparation of the magnetic separation reagent: dilute the 10 mg / mL magnetic microparticle concentrate to 1 mg / mL with magnetic separation reagent buffer, and equilibrate at 2-8℃ for 24 h; add 5 μg of acridine ester-labeled anti-ST2 antibody 2, the concentration of acridine ester-labeled anti-ST2 antibody 2 is 0.5 μg / mL, and mix at room temperature for 0.5 h; The magnetic separation reagent buffer has a pH of 7.4 and includes Tris (6.05 g / L), Proclin-300 (1.99 g / L), thimerosal (0.99 g / L), sodium chloride (8.98 g / L), bovine serum albumin (29.88 g / L), gelatin (9.86 g / L), N-ethylcis-butenedimide (1.05 g / L), Triton X-100 (0.98 g / L), Tween-20 (1.99 g / L), and the remainder is purified water. The calibrator buffer has a pH of 7.4 and consists of PBS (0.02 M), Casein (20 g / L), N-ethylcis-butenedimide (1.05 g / L), Proclin-300 (1.98 g / L), tetracycline (0.99 g / L), and purified water as the remaining component.

2. The magnetic microparticle chemiluminescence detection kit for rapid determination of soluble ST2 content according to claim 1, characterized in that, The kit contains six calibrators with different concentrations of ST2 antigen, two quality control samples with different concentrations of ST2 antigen, and the luminescent substrate solution is an acridine ester oxide substrate solution. The surface group of the magnetic particles is toluenesulfonyl group.

3. The magnetic particle chemiluminescence detection kit for rapid determination of soluble ST2 content according to claim 1, characterized in that, In the magnetic separation reagent, the mass ratio of anti-ST2 antibody 1 coated on the surface of magnetic microparticles to acridine ester-labeled anti-ST2 antibody 2 is 2 mg: 1 μg.

4. The magnetic microparticle chemiluminescence detection kit for rapid determination of soluble ST2 content according to claim 2, characterized in that, The concentration range of the calibrator is 0–300 ng / mL.

5. The magnetic microparticle chemiluminescence detection kit for rapid determination of soluble ST2 content according to claim 2, characterized in that, The six calibrators containing different concentrations of ST2 antigen were 0 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 150 ng / mL, and 300 ng / mL, respectively.

6. The magnetic microparticle chemiluminescence detection kit for rapid determination of soluble ST2 content according to claim 1, characterized in that, The blank limit of the kit is ≤0.0055 ng / mL, and the linear detection range of soluble growth-stimulating gene 2 protein is 1.0~300 ng / mL. The recovery rate of high-value serum with known soluble growth-stimulating gene 2 protein concentration was 90%~100% after being diluted 1:9 with low-value serum with known soluble growth-stimulating gene 2 protein concentration. No significant cross-reactivity was observed with 30 ng / mL troponin I or 30 ng / mL N-terminal pro-brain natriuretic peptide. There is no high-dose hook effect when the ST2 concentration is as high as 100,000 ng / mL; The intra-analytical precision was 1.34%, and the inter-analytical precision was 1.16%. It has strong anti-interference ability, and the determination of hemoglobin at 5 g / L, total bilirubin at 0.5 mmol / L, and fat emulsion at 10.0 g / L is not affected.

7. A method for preparing a magnetic microparticle chemiluminescence detection kit for rapid determination of soluble ST2 content according to any one of claims 1-6, for non-disease diagnostic purposes, comprising the following steps: Preparation of magnetic separation reagent: 1) Prepare coupling buffer, blocking solution, and storage solution. Wash and activate the magnetic beads, then wash, couple, and block the supernatant from the magnetic separation to obtain immunomagnetic beads coated with ST2 monoclonal antibody 1; 2) Adjust the volume of the immunomagnetic beads coated with ST2 monoclonal antibody 1 to a specific concentration using the storage solution of the magnetic separation reagent to obtain a concentrated magnetic microparticle solution; dilute the concentrated magnetic microparticle solution to a specific concentration using the magnetic separation reagent buffer; 3) Add 0.5... acridine ester-labeled anti-ST2 antibody 2 at g / mL, mixed at room temperature for 0.5 hours; Preparation of calibrators: 1) Prepare phosphate buffer according to the component content of the calibrator buffer; 2) Dissolve ST2 antigen in the calibrator buffer to prepare different concentrations; Add 20 μL of calibrator and 50 μL of magnetic separation reagent sequentially to the reaction tube, mix and incubate at 37 °C for 10 min; wash 3 times with washing buffer to remove unbound antibodies and impurities; 100 μL of each luminescent substrate excitation solution was added. After the acridinium ester catalyzed the luminescence of the substrate, the relative luminescence intensity (RLU) was measured using a chemiluminescence detector from Leadman Corporation to obtain a series of values ​​for ST2 concentration and luminescence value. A standard curve of soluble growth-stimulating gene 2 protein concentration-luminescence value was obtained by fitting the values ​​of the soluble growth-stimulating gene 2 protein concentration-luminescence value series. The concentration of the sample to be tested was calculated based on the fitted standard curve of soluble growth-stimulating gene 2 protein concentration-luminescence value. Within a certain range, RLU is directly proportional to the concentration of soluble growth-stimulating gene 2 protein antigen. The content of soluble growth-stimulating gene 2 protein in the test sample is read from the standard curve by interpolation.

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