A kit for rapid detection of sst2

Abstract

The application discloses a kit for rapidly detecting sST2, which comprises a test strip; the test strip comprises a back plate and a water absorption pad, an NC membrane, a fluorescent pad and a sample pad which are sequentially laid on the back plate; the fluorescent pad is coated with fluorescent microsphere labeled second monoclonal antibodies which are combined with the C-terminal unique region of sST2; the NC membrane is provided with a detection strip and a quality control strip, the detection strip is coated with first monoclonal antibodies which are combined with the 3rd immunoglobulin domain of sST2, and the quality control strip is coated with secondary antibodies which are combined with the second monoclonal antibodies; the kit can complete the rapid detection of sST2 within 6 minutes, effectively eliminates the cross reaction between isomers (such as soluble ST2 and transmembrane ST2), improves the detection accuracy and sensitivity, can directly use whole blood samples for detection, does not need to be centrifuged, saves the detection time, and can be applied to a full-automatic fluorescent immunoassay instrument, and automatic, convenient and rapid interpretation can be realized.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically to a kit for rapid detection of sST2. Background Technology

[0002] Heart failure (HF) is a disease caused by impaired cardiac pumping function, resulting in the heart's inability to meet the body's basal metabolic needs. Common triggers include myocardial infarction, cardiomyopathy, and myocarditis. High-risk groups include the elderly over 70 years of age, with an incidence rate exceeding 10% and a 5-year mortality rate reaching 50%. Heart failure can be classified according to the location of the lesion into left ventricular failure, right ventricular failure, and biventricular failure. For patients in high-risk stages, actively controlling risk factors, such as blood pressure, blood sugar, and blood lipids, as well as improving lifestyle and receiving necessary medication, can prevent or delay the onset and progression of heart failure. Simultaneously, for patients already exhibiting heart failure symptoms, timely medical attention and professional treatment are crucial.

[0003] Studies have shown that the Soluble Growth Stimulation Expressed Gene 2 (ST2), first identified by Tominaga et al. in the BALB / C-3T3 cell line in 1989, is a member of the IL-1 (interleukin-1) receptor family. This gene expresses two protein products: one with a transmembrane structure, called transmodel ST2 (ST2L); and one that can be secreted extracellularly, called secreted ST2 (sST2). Research has found that ST2 can be expressed by cardiac fibroblasts and cardiomyocytes, and is a cardiac protein induced by biomechanical stress. The ST2 gene is expressed in mast cells, activated helper T cells 2 (Th2), macrophages, and cardiomyocytes. The human ST2 gene is approximately 40 kb, located on human chromosome 2ql2, and encodes a soluble protein (sST2) and a transmembrane form of the protein (ST2L), the transcription of which is regulated by different promoters.

[0004] Currently, due to the existence of multiple isoforms of ST2 (such as transmembrane ST2 and different cleavage variants), false positive or false negative results are easily caused by antibody cross-binding during the detection process. Traditional ELISA or immunochromatographic methods mostly use single monoclonal antibodies, which cannot distinguish between isoforms, and the detection buffer is not optimized for isoform interference, resulting in insufficient sensitivity and specificity. In addition, ELISA technology for detecting ST2 has problems such as high requirements for detection equipment, high cost, many interfering factors, and poor repeatability. Traditional colloidal gold or fluorescence chromatography methods for detecting ST2 require 15-30 minutes, which cannot meet the needs of emergency and on-site testing. Therefore, how to develop rapid quantitative detection reagents has become an urgent problem to be solved. Summary of the Invention

[0005] The primary objective of this invention is to provide a kit for the rapid detection of sST2. This kit can complete the rapid detection of sST2 within 6 minutes. By optimizing antibody selection, reaction conditions, and blocking agent design, it effectively eliminates cross-reactivity between isomers (such as soluble ST2 and transmembrane ST2), improves detection accuracy and sensitivity, and solves the problem that there is currently no fluorescent immunochromatography that can complete direct detection of whole blood within 6 minutes with a CV < 10%.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0007] The first aspect of the present invention provides a kit for rapid detection of sST2, the kit comprising a test strip;

[0008] The test strip includes a backing plate and an absorbent pad, an NC membrane, a fluorescent pad, and a sample pad sequentially laid on the backing plate; the fluorescent pad is coated with a second monoclonal antibody labeled with fluorescent microspheres that bind to the unique C-terminal region of sST2.

[0009] The NC membrane has a detection band and a control band. The detection band is coated with a first monoclonal antibody that binds to the third immunoglobulin domain of sST2, and the control band is coated with a secondary antibody against the second monoclonal antibody.

[0010] Preferably, the kit further includes a blocking agent;

[0011] The blocking reagent is a buffer solution containing a polypeptide and Triton X-100, the amino acid sequence of which is shown in SEQ ID NO.1.

[0012] Preferably, the pH value of the buffer solution is 6.8~7.2; the content of the polypeptide is 10~50 μg / mL; and the content of Triton X-100 is 0.08%~0.12%.

[0013] Preferably, the kit further includes a blood filtration pad and / or a sample diluent; the blood filtration pad is disposed between the fluorescent pad and the sample pad, and its two ends overlap with the ends of the fluorescent pad and the sample pad, respectively.

[0014] Preferably, the sample diluent is a Tris solution containing 0.1% surfactant, wherein the concentration of the Tris solution is 0.1M and the pH value is 6.8~7.2; the surfactant is selected from any one of the Tween series surfactants.

[0015] Preferably, the fluorescent microsphere-labeled second monoclonal antibody is prepared by the following method:

[0016] (a) Add fluorescent microspheres to MES buffer, then add EDC and NHS and mix by shaking at room temperature to obtain a mixture;

[0017] (b) Centrifuge the mixture to remove the supernatant, then add BB buffer to reconstitute it, and then add the second monoclonal antibody and mix at room temperature to obtain the conjugated mixture;

[0018] (c) Centrifuge the conjugation mixture to remove the supernatant, add blocking solution to block, and then centrifuge to remove the supernatant to obtain the fluorescent microsphere-labeled second monoclonal antibody.

[0019] Preferably, the mass ratio of the fluorescent microspheres to the second monoclonal antibody is 1:(3~5).

[0020] Preferably, the sealing agent is a BSA solution with a concentration of 5% to 8%.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0022] This invention's kit enables rapid detection of sST2 within 6 minutes. By optimizing antibody selection, reaction conditions, and blocking agent design, it effectively eliminates cross-reactivity between isomers (such as soluble ST2 and transmembrane ST2), improving detection accuracy and sensitivity. Specifically, the interference rate of transmembrane ST2 is reduced to <5%, and the detection limit of sST2 reaches 5 pg / mL. Furthermore, this kit can be used directly with whole blood samples for detection without centrifugation, saving detection time. It is also compatible with fully automated fluorescence immunoassay analyzers, enabling automated, convenient, and rapid interpretation.

[0023] The coupling ratio of fluorescent microspheres to antibodies in this invention is controlled at 1:(3~5), which reduces steric hindrance, increases the chromatography rate, and shortens the detection time to 6 minutes, while maintaining high sensitivity and accuracy. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0025] Figure 1 This is a schematic diagram of the test strip structure in the kit for rapid detection of sST2 in the embodiments of the present invention;

[0026] Figure 2 This is a linear relationship graph of the test paper box detection when the coupling mass ratio is 1:2 in Experimental Example 1 of the present invention;

[0027] Figure 3 This is a linear relationship graph of the test paper box detection when the coupling mass ratio is 1:4 in Experimental Example 1 of the present invention;

[0028] Figure 4 This is a linear relationship graph of the test paper box detection when the coupling mass ratio is 1:6 in Experimental Example 1 of the present invention;

[0029] Figure 5 This is a comparative test result of the test reagent and the control reagent on 225 clinical serum samples in Experiment Example 4 of the present invention;

[0030] Figure 6 This is a comparative test result of the test reagent and the control reagent on 225 clinical plasma and serum samples in Experiment Example 4 of the present invention;

[0031] Figure 7 This is the comparative test result of the test reagent and the control reagent on 225 clinical whole blood and serum samples in Experiment Example 4 of the present invention.

[0032] In the attached diagram, 1-absorbent pad; 2-NC membrane; 3-backplate; 4-fluorescent pad; 5-blood filtration pad; 6-sample pad; 7-1-quality control strip; 7-2-detection strip. Detailed Implementation

[0033] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0034] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0035] This invention provides a kit for rapid detection of sST2, the kit comprising a test strip;

[0036] like Figure 1 As shown, the test strip includes a backing plate 3 and an absorbent pad 1, an NC membrane 2, a fluorescent pad 4, and a sample pad 6 sequentially laid on the backing plate 3; the fluorescent pad 4 is coated with a second monoclonal antibody labeled with fluorescent microspheres that bind to the unique C-terminal region of sST2.

[0037] The NC membrane 2 has a detection band 7-2 and a control band 7-1. The detection band 7-2 is coated with a first monoclonal antibody that binds to the third immunoglobulin domain of sST2, and the control band 7-1 is coated with a secondary antibody against the second monoclonal antibody.

[0038] This invention's kit enables rapid detection of sST2 within 6 minutes. By optimizing antibody selection, reaction conditions, and blocking agent design, it effectively eliminates cross-reactivity between isomers (such as soluble ST2 and transmembrane ST2), improving detection accuracy and sensitivity. Specifically, the interference rate of transmembrane ST2 is reduced to <5%, and the detection limit of sST2 reaches 5 pg / mL. Furthermore, this kit can be used directly with whole blood samples for detection without centrifugation, saving detection time. It is also compatible with fully automated fluorescence immunoassay analyzers, enabling automated, convenient, and rapid interpretation.

[0039] In one embodiment, the amino acid sequence position of the unique C-terminal region of sST2 is 201-220, as shown in SEQ ID NO:2, specifically LQASLAAAAAAAGGAAAAAA.

[0040] In one embodiment, the kit further includes a blocking agent;

[0041] The blocking reagent is a buffer containing a peptide and Triton X-100, and the amino acid sequence of the peptide is shown in SEQ ID NO.1, specifically GFSPPNISVQWYKNGKEVTLSHESVTQEDSVYTC.

[0042] In one embodiment, the pH value of the buffer solution is 6.8 to 7.2; the peptide content is 10 to 50 μg / mL; the Triton X-100 content is 0.08% to 0.12%; further, the buffer solution is a Tris buffer solution.

[0043] In one embodiment, the kit further includes a blood filtration pad 5 and / or a sample diluent; the blood filtration pad 5 is disposed between the fluorescent pad 4 and the sample pad 6, and its two ends overlap with the ends of the fluorescent pad 4 and the sample pad 6, respectively.

[0044] Furthermore, the blood filter pad 5 is made of glass fiber membrane.

[0045] In one embodiment, the sample diluent is a Tris solution containing 0.1% surfactant, wherein the Tris solution has a concentration of 0.1M and a pH value of 6.8 to 7.2; the surfactant is selected from any one of the Tween series surfactants.

[0046] In one embodiment, the fluorescent microsphere-labeled second monoclonal antibody is prepared by the following method:

[0047] (a) Add fluorescent microspheres to MES buffer, then add EDC and NHS and mix by shaking at room temperature to obtain a mixture;

[0048] (b) Centrifuge the mixture to remove the supernatant, then redissolve it in BB buffer. Next, add the second monoclonal antibody and mix well at room temperature to obtain a conjugate mixture.

[0049] (c) Centrifuge the conjugate mixture to remove the supernatant, then add the blocking solution for blocking. After centrifugation to remove the supernatant, the fluorescent microsphere-labeled second monoclonal antibody is obtained.

[0050] In one embodiment, the mass ratio of the fluorescent microspheres to the second monoclonal antibody can be 1∶(3 - 5).

[0051] The coupling ratio of the fluorescent microspheres to the antibody in the present invention is controlled at 1∶(3 - 5), reducing steric hindrance, improving the chromatography rate, shortening the detection time to 6 minutes, and at the same time maintaining high sensitivity and accuracy.

[0052] In one embodiment, the blocking agent is a BSA solution with a concentration of 5% - 8%.

[0053] The technical solution of the present invention will be further described in detail through specific examples below.

[0054] The full names of the following chemical formula abbreviations are as follows:

[0055] MES: English full name: 2-(N-Morpholino)ethanesulfonic acid; Chinese full name: 2-吗啉乙磺酸;

[0056] BB: English full name: Borate-Borax Buffer; Chinese full name: 硼酸-硼砂缓冲液;

[0057] EDC: English full name: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; Chinese full name: 1-乙基-3-(3-二甲基氨丙基)碳二亚胺;

[0058] NHS: English full name: N-Hydroxysuccinimide; Chinese full name: N-羟基琥珀酰亚胺;

[0059] BSA: English full name: Bovine Serum Albumin; Chinese full name: 牛血清白蛋白;

[0060] PB: English full name: Phosphate Buffer; Chinese full name: 磷酸盐缓冲液;

[0061] Tween 20: English full name: Polyoxyethylene (20) sorbitan monolaurate; Chinese full name:

[0062] Polyoxyethylene (20) sorbitan monolaurate; Example 1

[0063] This embodiment describes a method for preparing a sample pad, which includes the following steps:

[0064] (1) Preparation of sample pad solution:

[0065] Sodium caseinate, Tween 20, RBC, and sucrose were dissolved in 0.05M Tris-HCl pH7.0 solution to obtain a sample pad solution. The volume or mass-volume ratio of sodium caseinate, Tween 20, RBC, and sucrose dissolved in the Tris-HCl buffer was 0.1%, 0.2%, 0.3%, and 0.5%, respectively.

[0066] (2) Sample pad preparation:

[0067] Place the glass cellulose membrane (8964, Shanghai Jieyi Biotechnology Co., Ltd., size: 25cm*30cm) flat on a plastic pad. Use a pipette to add 40mL of sample pad solution per sheet, ensuring the liquid completely covers the sample pad. After initial drying, place it in a 37℃ oven to dry for 15 hours. After drying, place it in an aluminum foil bag, add desiccant, and seal it. The aluminum foil bag should be labeled with the name, process, batch number, quantity, and date. Store it at the intermediate station for later use.

[0068] Example 2

[0069] This embodiment describes a method for preparing a fluorescent pad, which includes the following steps:

[0070] I. Preparation of fluorescent microsphere-labeled second monoclonal antibody (microsphere to antibody mass ratio of 1:4):

[0071] (1) Activation: Take 25uL of fluorescent microspheres and add 975uL of 0.02M MES buffer, add EDC and NHS solution (final concentration is 25ug / mL), shake and mix at room temperature for 15min, and then centrifuge at 12000rpm for 15min.

[0072] (2) Coupling: After centrifugation, remove the supernatant from the microspheres, reconstitute them with 0.02M BB buffer to 1mL, add the second monoclonal antibody (antibody with clone code 10C2 from Wuhan Aoke Biotechnology Co., Ltd.) to a final concentration of 20ug / mL, and mix at room temperature for 4 hours;

[0073] (3) Blocking: Centrifuge the conjugate at 12000 rpm for 15 min, add 1 mL of blocking solution (5% BSA) and mix at room temperature for 1 hour, then centrifuge at 12000 rpm for 15 min;

[0074] (4) Reconstitution: After centrifugation, remove the supernatant from the microspheres and reconstitute them to a volume of 1 mL using a reconstitution solution (0.05 M PB solution);

[0075] II. Preparation of fluorescent pads:

[0076] Spray the reconstituted solution in (4) at 2 μL / cm onto the pad and bake at 45℃ for 60 min to obtain the fluorescent pad. The RATE value in the pad spraying process is 4 µL / cm and the air pressure is 2 psi. When there are breaks or uneven thickness, use a pen to draw lines.

[0077] Example 3

[0078] This embodiment describes a method for preparing an NC membrane, which includes the following steps:

[0079] (1) Preparation of coating solution:

[0080] Na2HPO4∙12H2O, K2HPO4∙2H2O, and sucrose were dissolved in water to obtain a coating solution, wherein the concentration of Na2HPO4∙12H2O was 0.05 mol, the concentration of K2HPO4∙2H2O was 0.05 mol, and the mass-volume concentration of sucrose was 2% (g / ml).

[0081] (2) Preparation of the coating solution for the detection strips:

[0082] The first monoclonal antibody that binds to the third immunoglobulin domain of sST2 (the antibody with clone code 3C4 from Wuhan Aoke Biotechnology Co., Ltd.) was added to the coating solution to obtain a detection band coating solution with a concentration of 0.5 mg / ml.

[0083] (3) Preparation of coating solution for quality control strips:

[0084] The secondary antibody against the second monoclonal antibody (goat anti-mouse, from Luoyang Baitong Experimental Materials Center) was added to the coating solution to obtain a quality control strip coating solution with a concentration of 0.5 mg / ml.

[0085] (4) The distance between the C and T lines is 6.0 mm, and the RATE value is 1.0 μL / cm. Place the NC membrane on the X platform, and place it along the upper edge of the paper card on the X platform. If there are any breaks or abnormal line thicknesses in the middle when applying the membrane, mark them with a pen. Before the membrane application is finished, if the antibody cannot fill the entire tubing, add ultrapure water to the corresponding membrane application pump to replenish the tubing. After applying the membrane, indicate the upper and lower positions of the C and T lines, the product name, and the date, and move it to a clean tray. When picking up and putting down the NC membrane, only touch the two ends to avoid damaging the NC membrane.

[0086] (5) Place the dotted NC film in a 45℃ oven and dry for 15 hours; when handling the film, only touch the two ends of the film to avoid damaging the middle part; after drying, put it in an aluminum foil bag, add desiccant, and seal it. The aluminum foil bag should be marked with the name, process, batch number, quantity, and date; keep it at the intermediate station for later use.

[0087] Example 4

[0088] This embodiment describes a method for preparing an inhibitor, which includes the following steps:

[0089] The peptide and Triton X-100 were added to a buffer solution to obtain an inhibitor, wherein the amino acid sequence of the peptide is shown in SEQ ID NO.1, specifically GFSPPNISVQWYKNGKEVTLSHESVTQEDSVYTC; the pH of the buffer solution is 7; the peptide concentration is 20 μg / mL; the Triton X-100 concentration is 0.1%; and the buffer solution is Tris buffer.

[0090] Example 5

[0091] This embodiment is a kit for rapid detection of sST2, the kit including test strips;

[0092] like Figure 1 As shown, the test strip includes a backing plate 3 and an absorbent pad 1, an NC membrane 2 (Example 3), a fluorescent pad 4 (Example 2), and a sample pad 6 (Example 1) sequentially laid on the backing plate 3.

[0093] Example 6

[0094] This embodiment is a kit for rapid detection of sST2, the kit including test strips, blocking reagents and sample diluents;

[0095] like Figure 1 As shown, the test strip includes a backing plate 3 and an absorbent pad 1, an NC membrane 2 (prepared by the method of Example 3), a fluorescent pad 4 (prepared by the method of Example 2), a blood filter pad 5, and a sample pad 6 (prepared by the method of Example 1) sequentially laid on the backing plate 3.

[0096] The blocking agent is the blocking agent prepared in Example 4;

[0097] The sample diluent was a Tris solution containing 0.1% surfactant, wherein the Tris solution concentration was 0.1M and the pH value was 7; the surfactant was Tween-60.

[0098] After the test strip in the kit of this invention detects color development, the fluorescence intensity is read by a fully automated fluorescence immunoassay analyzer, and the concentration of sST2 in the sample is determined according to the standard curve of fluorescence intensity and sST2 concentration.

[0099] Experimental Example 1

[0100] This experimental example studies the effect of different conjugation ratios of fluorescent microsphere-labeled second monoclonal antibodies on the detection performance of the kit.

[0101] Creating a standard curve:

[0102] Serial dilution of standards: The target antigen standards were prepared with fetal bovine serum to obtain standards with concentrations of 5 ng / mL, 10 ng / mL, 25 ng / mL, 40 ng / mL, 100 ng / mL, and 200 ng / mL.

[0103] Test standard: Add the standard of each concentration to the test strip, and after the reaction, read the fluorescence intensity (FI) of the detection line (T line) with a fluorescence reader;

[0104] Data processing: With the standard concentration as the X-axis and the corresponding FI value as the Y-axis, a four-parameter Logistic model (Y=A+(DA) / (1+(X / C)^B)) was used to fit the curve, ensuring that R²>0.98. The specific linear equation is y=20.892125+(0.25725220.892125) / (1+(1776.909031)^1.668311).

[0105] Validation: Verify the accuracy of the curve using quality control materials, and save the equation for quantification of unknown samples.

[0106] Following the preparation method in Example 2, fluorescent pads with a mass ratio of 1:2, 1:4, and 1:6 were prepared by labeling fluorescent microspheres with a second monoclonal antibody conjugated to them.

[0107] Then, the fluorescent pads described above were used to prepare kits for rapid detection of sST2 according to Example 6;

[0108] Sample solutions of sST2 at concentrations of 50, 100, and 200 ng / ml were prepared and detected using the above kit. Ten replicates were performed, and the mean and standard deviation were calculated. The CV value was also calculated. The results are shown in Table 1.

[0109] Table 1

[0110] Standard solutions of sST2 with concentrations of 5, 10, 25, 40, 100, and 200 ng / ml were prepared and detected using the above kit. Three replicates were performed, the average value was calculated, and linearity analysis was conducted. The results are shown in Tables 2 and 3.

[0111] Table 2

[0112] Table 3

[0113] During the above detection process, the liquid migration speed was calculated and the average value was calculated. The results are shown in Table 4.

[0114] Table 4

[0115] As shown in Tables 1 to 4, when the mass ratio of microspheres to antibody conjugation in this embodiment is 1:4, the detection CV is the lowest, the linear correlation coefficient is the highest, and the liquid migration speed is the fastest.

[0116] Experiment Example 2

[0117] This experimental example compares the test results of the reagent kit of Example 6 of this invention with those of a comparative manufacturer's reagent kit.

[0118] The signal values ​​of a 50 ng / mL sample were detected at 6 minutes and 10 minutes using the present invention and the control reagent (soluble growth-stimulating gene 2 protein assay reagent (fluorescent immunochromatography) produced by Guangzhou Wondfo Biotech Co., Ltd.). The values ​​were measured 10 times and the average value was calculated. The lower the CV, the better the stability.

[0119] The experimental results are shown in Table 5:

[0120] Table 5

[0121] As shown in Table 5, the CV of the detection results of the present invention at 6 minutes and 10 minutes are lower than those of the control reagent, indicating that its stability is higher than that of the control reagent.

[0122] Experimental Example 3

[0123] This experimental example studies the effect of different concentrations of blocking agent on the detection performance of the reagent kit;

[0124] Fluorescent pads were prepared using 2.5%, 5%, and 8% BSA solutions as blocking agents, respectively, according to the preparation method in Example 2.

[0125] Then, the fluorescent pads described above were used to prepare kits for rapid detection of sST2 according to Example 6;

[0126] Sample solutions of sST2 at concentrations of 50, 100, and ng / ml were prepared and tested using the above kit. Ten replicates were performed, and the mean and standard deviation were calculated. The CV value was also calculated. The results are shown in Table 6.

[0127] Table 6

[0128] As shown in Table 6, when the BSA concentration is greater than or equal to 5%, the background is clean and the chromatography effect is good. Therefore, the BSA concentration of 5% was selected.

[0129] Experiment Example 4

[0130] This experimental example is a comparative study of the clinical testing performance of the kit prepared in Example 6 with existing commercially available products.

[0131] Using the kit described in this application as the test reagent in a clinical evaluation trial (denoted as the assessment reagent), and selecting an approved ST2 detection kit as the control reagent (soluble growth-stimulating gene 2 protein assay reagent (fluorescent immunochromatography) produced by Guangzhou Wondfo Biotech Co., Ltd.), 225 clinical samples (serum) were tested, and the results are as follows: Figure 5 As shown;

[0132] The kit described in this application was used as the test reagent in a clinical evaluation trial (denoted as the test reagent) to test 225 clinical samples (plasma and serum). The results are as follows: Figure 6 As shown;

[0133] The kit described in this application was used as the test reagent in a clinical evaluation trial (denoted as the test reagent) to test 225 clinical samples (whole blood and serum). The results are as follows: Figure 7 As shown;

[0134] Depend on Figures 5-7 It can be known that:

[0135] The test results of the product evaluated in this invention show good consistency with those of the comparative product, and the two systems are equivalent. Detection results of homologous plasma, whole blood, and serum show good consistency among different sample types. Therefore, the product of this invention is suitable for the in vitro quantitative detection of the content of soluble growth-stimulating gene 2 protein in human whole blood, serum, and plasma, and is applicable to clinical testing.

[0136] Experimental Example 5

[0137] This experimental example is for screening peptides in blocking agents:

[0138] Target analysis: The extracellular segment sequence of tmST2 was analyzed to identify key epitopes that bind to interfering antibodies and exclude soluble ST2 homologous regions;

[0139] Peptide library design: Synthesize a peptide library covering epitopes (containing the precursor sequence of SEQ ID NO:1);

[0140] Initial screening: High-affinity peptides (KD < 10⁻) are screened using ELISA. 7 M);

[0141] Blocking validation: Competition experiments showed that the candidate peptides (10-50 μg / mL) could dose-dependently block the binding of interfering antibodies to natural tmST2 (inhibition rate >90%) without affecting the recognition of the target antibody;

[0142] Specificity confirmation: No cross-reactivity was found, and SEQ ID NO:1 (GFSPPNISVQWYKNGKEVTLSHESVTQEDSVYTC) was ultimately determined to be the optimal blocking agent.

[0143] Experimental Example 6

[0144] This experimental example is a screening experiment for the first and second monoclonal antibodies:

[0145] Four candidate antibodies (Ab1, Ab2, Ab3, Ab4) were tested using a matrix method.

[0146] Ab3: Targets mouse ST2L, with no cross-reactivity with rat and human ST2L (from R&D Systems, rat-derived monoclonal antibody, clone code DJ8); Ab4: A unique sequence targeting the sST2 cleavage site (from MD Bioproducts rat anti-human sST2 monoclonal antibody, clone code 10102).

[0147] The antibody pairs with the strongest signals and the best specificity were selected, and the antibody combinations are shown in Table 7:

[0148] Table 7

[0149] Screening was conducted based on a T-line fluorescence intensity (FI) > 2000 (strong signal), a T-line FI < 20 (<1%) in the ST2L extracellular segment cross-reactivity group, and normal C-line color development. The Ab1 / Ab2 combination was tested using a matrix method to select the antibody pair with the strongest signal and optimal specificity.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A kit for rapid detection of sST2, characterized in that, The kit includes test strips; The test strip includes a backing plate and an absorbent pad, an NC membrane, a fluorescent pad, and a sample pad sequentially laid on the backing plate; the fluorescent pad is coated with a second monoclonal antibody labeled with fluorescent microspheres that bind to the unique C-terminal region of sST2. The NC membrane has a detection band and a control band. The detection band is coated with a first monoclonal antibody that binds to the third immunoglobulin domain of sST2, and the control band is coated with a secondary antibody against the second monoclonal antibody. The kit also includes blocking agents; The blocking reagent is a buffer solution containing a polypeptide and Triton X-100, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The kit for rapid detection of sST2 according to claim 1, characterized in that, The pH of the buffer solution is 6.8 to 7.2; the peptide content is 10 to 50 μg / mL; and the Triton X-100 content is 0.08% to 0.12%.

3. The kit for rapid detection of sST2 according to claim 1, characterized in that, The kit also includes a blood filtration pad and / or a sample diluent; the blood filtration pad is disposed between the fluorescent pad and the sample pad, and its two ends overlap with the ends of the fluorescent pad and the sample pad, respectively.

4. The kit for rapid detection of sST2 according to claim 3, characterized in that, The sample diluent is a Tris solution containing 0.1% surfactant, the concentration of which is 0.1M and the pH value is 6.8-7.2; the surfactant is selected from any one of the Tween series surfactants.

5. The kit for rapid detection of sST2 according to claim 1, characterized in that, The fluorescent microsphere-labeled second monoclonal antibody was prepared by the following method: (a) Add fluorescent microspheres to MES buffer, then add EDC and NHS and mix by shaking at room temperature to obtain a mixture; (b) Centrifuge the mixture to remove the supernatant, then add BB buffer to reconstitute it, and then add the second monoclonal antibody and mix at room temperature to obtain the conjugated mixture; (c) Centrifuge the conjugation mixture to remove the supernatant, add blocking solution to block, and then centrifuge to remove the supernatant to obtain the fluorescent microsphere-labeled second monoclonal antibody.

6. The kit for rapid detection of sST2 according to claim 5, characterized in that, The mass ratio of the fluorescent microspheres to the second monoclonal antibody is 1:(3~5).

7. The kit for rapid detection of sST2 according to claim 5, characterized in that, The sealing solution is a BSA solution with a concentration of 5% to 8%.

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