Kit for rapidly detecting sST2

Abstract

The invention discloses a kit for rapidly detecting sST2. The kit 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 a second monoclonal antibody which is labeled by fluorescent microspheres and is combined with a 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 a first monoclonal antibody combined with a third immunoglobulin structural domain of sST2, and the quality control strip is coated with a second antibody for resisting a second monoclonal antibody; according to the kit, rapid detection of sST2 can be completed within 6 minutes, cross reaction between isomers (such as soluble ST2 and transmembrane type ST2) is effectively eliminated, the detection accuracy and sensitivity are improved, the kit can directly use a whole blood sample for detection, centrifugation is not needed, and the detection time is saved; and the method is suitable for a full-automatic fluorescence immunoassay analyzer, and can realize automatic, convenient and rapid interpretation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a kit for rapidly detecting sST2. BACKGROUND

[0002] Heart failure (HF) is a disease caused by abnormal heart pumping function, which leads to the heart failing to meet the basic metabolic needs of the whole body. Common causes include myocardial infarction, cardiomyopathy, myocarditis, etc. The high-risk population includes people over 70 years old, and the incidence rate is more than 10%, and the 5-year mortality rate can reach 50%. Heart failure can be divided into left heart failure, right heart failure and whole heart failure according to the lesion site. For patients at high risk, actively controlling risk factors such as blood pressure, blood sugar, blood lipids, and improving lifestyle and receiving necessary drug treatment can prevent or delay the occurrence and development of heart failure. At the same time, for patients who have already appeared heart failure symptoms, it is also very important to seek medical treatment and receive professional treatment in time.

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

[0004] At present, due to the existence of various 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 detection; traditional ELISA or immunochromatography method uses a single monoclonal antibody, which cannot distinguish isoforms, and the detection buffer is not optimized for isoform interference, resulting in insufficient sensitivity and specificity; in addition, the ELISA technology for detecting ST2 has the problems of high requirement for detection equipment, high cost, many interference factors and poor repeatability; the traditional colloidal gold or fluorescent chromatography method for detecting ST2 needs 15-30 minutes, which cannot meet the needs of emergency and on-site detection, so how to make a rapid quantitative detection reagent becomes an urgent problem to be solved. SUMMARY

[0005] The first object of the present application is to provide a kit for rapid detection of sST2, which can complete the rapid detection of sST2 within 6 minutes, effectively eliminate the cross-reaction between isomers (such as soluble ST2 and transmembrane ST2) by optimizing the selection of antibodies, reaction conditions and the design of blocking agents, improve the detection accuracy and sensitivity, and solve the problem that there is no fluorescence immunoassay that can complete the direct detection of whole blood within 6 minutes and has a CV of less than 10%.

[0006] In order to achieve the above object of the present application, the following technical solutions are adopted: The first aspect of the present application provides a kit for rapid detection of 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 that bind to the C-terminal unique region of sST2; The NC membrane has a detection band and a quality control band, the detection band is coated with first monoclonal antibodies that bind to the 3rd immunoglobulin domain of sST2, and the quality control band is coated with secondary antibodies against the second monoclonal antibodies.

[0007] Preferably, the kit further comprises a blocking reagent; The blocking reagent is a buffer solution containing a polypeptide and Triton X-100, and the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1.

[0008] 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%.

[0009] Preferably, the kit further comprises a blood filter pad and / or a sample diluent; the blood filter pad is arranged between the fluorescent pad and the sample pad, and the two ends thereof are respectively overlapped with the end of the fluorescent pad and the end of the sample pad.

[0010] Preferably, the sample diluent is a Tris solution containing 0.1% surfactant, the concentration of the Tris solution is 0.1M, and the pH value is 6.8-7.2; and the surfactant is selected from any one of the Tween surfactants.

[0011] Preferably, the fluorescent microsphere-labeled second monoclonal antibodies are prepared by the following method: (a) adding fluorescent microspheres to a MES buffer solution, then adding EDC and NHS and mixing at room temperature to obtain a mixed solution; (b) centrifuging the mixed solution to remove the supernatant, redissolving the mixed solution by adding BB buffer, then adding the second monoclonal antibody to mix at room temperature to obtain a coupling mixture; (c) centrifuging the coupling mixture to remove the supernatant, adding a blocking solution to block, then removing the supernatant by centrifugation to obtain the fluorescent microsphere-labeled second monoclonal antibody.

[0012] Preferably, the mass ratio of the fluorescent microspheres and the second monoclonal antibody is 1: (3-5).

[0013] Preferably, the blocking agent is a BSA solution with a concentration of 5%-8%.

[0014] Compared with the prior art, the present application has at least the following beneficial effects: The kit of the present application can complete the rapid detection of sST2 within 6 minutes. By optimizing the selection of antibodies, reaction conditions and the design of blocking agents, the cross-reaction between isomers (such as soluble ST2 and transmembrane ST2) is effectively eliminated, the detection accuracy and sensitivity are improved, specifically, the interference rate of transmembrane ST2 is reduced to <5%, and the lower limit of sST2 detection is 5 pg / mL. In addition, the kit of the present application can directly use whole blood samples for detection without centrifugation, saving detection time. It can be applied to a fully automatic fluorescent immunoassay instrument, and automatic, convenient and rapid interpretation can be realized.

[0015] The coupling ratio of the fluorescent microspheres and the antibodies in the present application is controlled at 1: (3-5), which reduces the steric hindrance, improves the chromatography rate, shortens the detection time to 6 minutes, and at the same time maintains high sensitivity and accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0017] Figure 1 Figure 1 is a structural schematic diagram of a test strip in the kit for rapidly detecting sST2 in the embodiments of the present application; Figure 2 Figure 2 is a linear relationship diagram of the test strip in the kit when the coupling mass ratio is 1:2 in Experimental Example 1 of the present application; Figure 3 Figure 3 is a linear relationship diagram of the test strip in the kit when the coupling mass ratio is 1:4 in Experimental Example 1 of the present application; Figure 4 Figure 4 is a linear relationship diagram of the test strip in the kit when the coupling mass ratio is 1:6 in Experimental Example 1 of the present application; Figure 5This 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; 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; 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.

[0018] 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

[0019] 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.

[0020] 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.

[0021] This invention provides a kit for rapid detection of sST2, the kit comprising a test strip; 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. 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.

[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] In an embodiment, the amino acid sequence of the C-terminal unique region of sST2 is at positions 201-220 of SEQ ID NO: 2, specifically LQASLAAAAAAAGGAAAAAA.

[0024] In an embodiment, the kit further comprises a blocking reagent. The blocking reagent is a buffer solution containing a polypeptide and Triton X-100, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1, specifically GFSPPNISVQWYKNGKEVTLSHESVTQEDSVYTC.

[0025] In an embodiment, the pH value of the buffer solution is 6.8-7.2; the polypeptide content is 10-50 μg / mL; the content of Triton X-100 is 0.08%-0.12%; further, the buffer solution is Tris buffer solution.

[0026] In an embodiment, the kit further comprises a blood filter pad 5 and / or a sample diluent; the blood filter pad 5 is arranged between the fluorescent pad 4 and the sample pad 6, and the two ends are respectively overlapped with the end of the fluorescent pad 4 and the sample pad 6.

[0027] Further, the material of the blood filter pad 5 is glass fiber membrane.

[0028] In an embodiment, the sample diluent is a Tris solution containing 0.1% surfactant, wherein the concentration of the Tris solution is 0.1 M, the pH value is 6.8-7.2; and the surfactant is selected from any one of the Tween surfactants.

[0029] In an embodiment, the fluorescent microsphere-labeled second monoclonal antibody is prepared by the following method: (a) adding fluorescent microspheres to a MES buffer solution, then adding EDC and NHS and mixing at room temperature, to obtain a mixed solution; (b) centrifuging the mixed solution to remove the supernatant, then adding a BB buffer solution for re-dissolution, then adding the second monoclonal antibody and mixing at room temperature, to obtain a coupling mixture; (c) centrifuging the coupling mixture to remove the supernatant, then adding a blocking solution for blocking, then removing the supernatant by centrifugation, to obtain the fluorescent microsphere-labeled second monoclonal antibody.

[0030] In an embodiment, the mass ratio of the fluorescent microspheres and the second monoclonal antibody can be 1: (3-5).

[0031] The coupling ratio of the fluorescent microspheres and the antibody in the application is controlled at 1: (3-5), the steric hindrance is reduced, the chromatography rate is improved, the detection time is shortened to 6 minutes, and high sensitivity and accuracy are maintained.

[0032] In an embodiment, the blocking agent is a BSA solution with a concentration of 5%-8%.

[0033] The technical solutions of the application are further described in detail through specific embodiments.

[0034] The following chemical formulae are abbreviated as follows: MES: English full name: 2-(N-Morpholino)ethanesulfonic acid; Chinese full name: 2-morpholinoethanesulfonic acid; BB: English full name: Borate-Borax Buffer; Chinese full name: Borate-Borax Buffer; EDC: English full name: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; Chinese full name: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS: English full name: N-Hydroxysuccinimide; Chinese full name: N-hydroxysuccinimide; BSA: English full name: Bovine Serum Albumin; Chinese full name: Bovine Serum Albumin; PB: English full name: Phosphate Buffer; Chinese full name: Phosphate Buffer; Tween 20: English full name: Polyoxyethylene (20) sorbitan monolaurate; Chinese full name: Polyoxyethylene (20) sorbitan monolaurate; Example 1

[0035] This embodiment is a preparation method of a sample pad, which comprises the following steps: (1) Preparation of sample pad solution: Sodium caseinate, Tween 20, RBC, and sucrose are respectively dissolved in a 0.05M Tris-HCl pH7.0 solution to obtain a sample pad solution, wherein the volume or mass volume ratio of sodium caseinate, Tween 20, RBC, and sucrose dissolved in the Tris-HCl buffer is 0.1%, 0.2%, 0.3%, and 0.5%, respectively.

[0036] (2) Sample pad preparation: Place the glass cellulose membrane (8964, Shanghai Jieyi Biotechnology Co., Ltd., size: 25 cm*30 cm) on a plastic pad, and use a pipette to add 40 mL of sample pad solution per piece, and the liquid completely soaks the sample pad; after preliminary drying, place it in a 37°C oven for drying, and dry for 15 hours; after drying, place it in an aluminum foil bag, add a drying agent, and seal; the aluminum foil bag should be marked with the name, process, batch number, quantity, and date; and place it in the middle station for standby use.

[0037] Example 2 The present embodiment is a preparation method of a fluorescent pad, which comprises the following steps: I. Preparation of fluorescent microsphere-labeled second monoclonal antibody (mass ratio of microspheres to antibody is 1:4): (1) Activation: Take 25 uL of fluorescent microspheres and add 975 uL of 0.02M MES buffer solution, and add EDC and NHS solution (final concentration is 25 ug / mL), shake and mix at room temperature for 15 min, and then centrifuge at 12000 rpm for 15 min; (2) Coupling: Remove the supernatant of the centrifuged microspheres, and resuspend to 1 mL using 0.02M BB buffer solution, and add second monoclonal antibody (antibody from Wuhan Aoke Biological Technology Co., Ltd., clone code is 10C2) with a final concentration of 20 ug / mL, and mix at room temperature for 4 hours; (3) Blocking: Centrifuge the coupling product at 12000 rpm for 15 min, add 1 mL of blocking solution (5% BSA) and mix at room temperature for 1 hour, and then centrifuge at 12000 rpm for 15 min; (4) Resuspension: Remove the supernatant of the centrifuged microspheres, and resuspend to 1 mL volume using resuspension solution (0.05M PB solution); II. Preparation of fluorescent pad: Spray the resuspension solution in (4) at 2 uL / cm on the pad, and dry at 45°C for 60 min to obtain the fluorescent pad, wherein the RATE value in the pad spraying process is 4 uL / cm, and the air pressure is 2 psi; use a pen line when a breakpoint or uneven thickness occurs.

[0038] Example 3 The present embodiment is a preparation method of an NC membrane, which comprises the following steps: (1) Preparation of coating solution: Dissolve Na2HPO4·12H2O, K2HPO4·2H2O, and sucrose in water to obtain a coating solution, wherein the concentration of Na2HPO4·12H2O is 0.05 mol, the concentration of K2HPO4·2H2O is 0.05 mol, and the mass concentration of sucrose is 2% (g / ml).

[0039] (2) Preparation of detection strip coating solution: A first monoclonal antibody binding the third immunoglobulin domain of sST2 (antibody from Wuhan Aoke Biological Technology Co., Ltd. with clone code 3C4) is added to the coating solution to obtain a detection strip coating solution with a concentration of 0.5 mg / ml; (3) Preparation of quality control strip coating solution: A secondary antibody (goat anti-mouse from Luoyang Baoao Tong Experimental Material Center) against the second monoclonal antibody is added to the coating solution to obtain a quality control strip coating solution with a concentration of 0.5 mg / ml. (4) The distance between the C and T lines is 6.0 mm, and the RATE value is 1.0 μL / cm. When placing the NC membrane on the X platform, it is placed along the paper card upper edge of the X platform. If a breakpoint appears in the middle or the line thickness is abnormal during the dotting process, a pen is used to draw a line on the NC membrane to mark it. If the antibody cannot fill the entire pipeline before the dotting process is completed, ultrapure water should be added to the corresponding dotting pump to supplement the pipeline. After dotting the membrane, the upper and lower positions of the C and T lines, the product name, and the date should be noted, and the NC membrane should be moved to a clean cradle. When taking and placing the NC membrane, only the two ends should be touched to avoid damaging the NC membrane. (5) The dotted NC membrane is placed in a 45°C oven for 15 h. When taking and placing the membrane, only the two ends of the membrane should be touched to avoid damaging the middle part of the membrane. After drying, it is placed in an aluminum foil bag, a drying agent is added, and it is sealed. The name, process, batch number, quantity, and date should be noted on the aluminum foil bag. It is placed in the middle station for standby use.

[0040] Example 4 The present embodiment is a preparation method of a blocking agent, which comprises the following steps: A polypeptide and Triton X-100 are added to a buffer solution to obtain a blocking agent, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1, specifically GFSPPNISVQWYKNGKEVTLSHESVTQEDSVYTC; the pH value of the buffer solution is 7; the content of the polypeptide is 20 μg / mL; the content of Triton X-100 is 0.1%; and the buffer solution is a Tris buffer solution.

[0041] Example 5 The present embodiment is a kit for rapidly detecting sST2, which comprises a test strip. As shown in Figure 1 The test strip comprises a back plate 3 and a water absorption pad 1, an NC membrane 2 (Example 3), a fluorescent pad 4 (Example 2), and a sample pad 6 (Example 1) laid in sequence on the back plate 3.

[0042] Example 6 The present embodiment is a kit for rapidly detecting sST2, which comprises a test strip, a blocking reagent, and a sample diluent. As shown in Figure 1 The test strip includes a back plate 3 and a water absorption pad 1, an NC film 2 (prepared by the method of Example 3), a fluorescent pad 4 (prepared by the method of Example 2), a blood filtration pad 5 and a sample pad 6 (prepared by the method of Example 1) laid on the back plate 3 in sequence; The blocking reagent is the blocking reagent prepared in Example 4; The sample diluent is a Tris solution containing 0.1% surfactant, the Tris solution has a concentration of 0.1M and a pH value of 7; and the surfactant is Tween-60.

[0043] After the test strip in the kit of the present application is detected for color development, the fluorescence intensity is read by a full-automatic fluorescence immunoassay analyzer, and then the concentration of sST2 in the sample is determined according to the standard curve of fluorescence intensity and sST2 concentration.

[0044] Experimental Example 1 This experimental example is a study on the influence of different coupling ratios of fluorescent microsphere-labeled second monoclonal antibodies on the detection performance of the kit; Preparation of standard curve: Gradient dilution of standard: the target antigen standard is prepared into standard solutions with concentrations of 5 ng / mL, 10 ng / mL, 25 ng / mL, 40 ng / mL, 100 ng / mL and 200 ng / mL by using calf serum.

[0045] Detection of standard: each concentration of standard is added to the test strip, and after reaction, the fluorescence intensity (FI) of the detection line (T line) is read by a fluorescence reader; Data processing: the concentration of the standard is taken as the X axis, and the corresponding FI value is taken as the Y axis, a four-parameter Logistic model (Y=A+(D-A) / (1+(X / C)^B)) is used to fit the curve, and R²>0.98 is ensured, and the linear equation is y=20.892125+(0.25725220.892125) / (1+(1776.909031)^1.668311.

[0046] Verification: the accuracy of the curve is verified by using a quality control product, and the equation is saved for the quantification of unknown samples.

[0047] According to the preparation method in Example 2, fluorescent pads with fluorescent microsphere-labeled second monoclonal antibodies with coupling mass ratios of 1:2, 1:4 and 1:6 are prepared respectively; Then, the above fluorescent pads are respectively prepared into kits for rapid detection of sST2 according to Example 6; Sample solutions of sST2 with concentrations of 50 ng / ml, 100 ng / ml and 200 ng / ml are prepared, and the above kits are used for detection, 10 repeats are performed, the average value and the standard deviation are calculated, and the CV value is calculated, and the results are shown in Table 1. Table 1

[0048] The standard solution of 5, 10, 25, 40, 100, 200 ng / ml of sST2 was prepared and detected by using the above kit, 3 repeats, the average value was calculated, and the linear relationship was analyzed, and the results are shown in Table 2 and Table 3; Table 2

[0049] Table 3

[0050] In the above detection process, the liquid migration speed was calculated, and the average value was calculated, and the results are shown in Table 4;

[0051] Table 4

[0052] From Tables 1 to 4, it can be seen that when the mass ratio of the microspheres and the antibody in the 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.

[0053] Experimental Example 2 This experimental example is a comparison of the detection results of the kit of Example 6 of the application and the kit of a comparative manufacturer.

[0054] The signal values of the sample with a concentration of 50 ng / mL were detected by using the application and the comparative reagent (soluble growth stimulating expression gene 2 protein assay reagent (fluorescence immuno-chromatography) produced by Guangzhou Wanfu Biotechnology Co., Ltd.) at 6 minutes and 10 minutes, respectively, and the average value was measured for 10 times. The lower the CV, the better the stability.

[0055] The experimental results are shown in Table 5: Table 5

[0056] From Table 5, it can be seen that the CV of the detection results of the application at 6 minutes and 10 minutes is lower than that of the comparative reagent, and it is considered that the stability of the application is higher than that of the comparative reagent.

[0057] Experimental Example 3 This experimental example is a study on the influence of the detection performance of the kit prepared by different concentrations of blocking agents; According to the preparation method in Example 2, 2.5%, 5%, and 8% BSA solutions were used as blocking agents to prepare fluorescent pads; Then, the above fluorescent pads were made into kits for rapid detection of sST2 according to Example 6; Prepare a sample solution of 50, 100, ng / ml of sST2, and detect using the kit described above, 10 repeats, calculate the average and standard deviation, and calculate the CV value, and the results are shown in Table 6; Table 6

[0058] From Table 6, it can be seen that when the BSA concentration is greater than or equal to 5%, the background is clean and the chromatographic effect is good, so the concentration of BSA is selected to be 5%.

[0059] Experimental Example 4 This experimental example is a comparative study of the clinical detection performance of the reagent kit prepared in Example 6 and the existing commercially available product; The kit of the present application is used as the reagent to be evaluated in the clinical evaluation test (denoted as the evaluation reagent), and the ST2 detection kit approved for marketing (soluble growth stimulating expression gene 2 protein detection reagent (fluorescence immunochromatography method) produced by Guangzhou Wanfu Biotechnology Co., Ltd.) is used as the comparative reagent. 225 clinical samples (serum) are detected, and the results are shown in Table 7. Figure 5 The kit of the present application is used as the reagent to be evaluated in the clinical evaluation test (denoted as the evaluation reagent) to detect 225 clinical samples (plasma, serum), and the results are shown in Table 8. Figure 6 The kit of the present application is used as the reagent to be evaluated in the clinical evaluation test (denoted as the evaluation reagent) to detect 225 clinical samples (plasma, serum), and the results are shown in Table 8. Figure 7 It can be seen from Table 8 that: Figures 5-7 The detection results of the evaluation product and the comparative product have good consistency, and the two systems are equivalent. The detection results of the homologous plasma, whole blood and serum show that the detection results of different sample types have good consistency. Therefore, the product of the present application is used for in vitro quantitative detection of the content of soluble growth stimulating expression gene 2 protein in human whole blood, serum and plasma, and is suitable for clinical detection.

[0060] Experimental Example 5 This experimental example is the screening of polypeptides in the blocking agent: Target analysis: analyze the extracellular segment sequence of tmST2, lock the key epitope combined with the interference antibody, and exclude the soluble ST2 homologous region; Peptide library design: synthesize a polypeptide library covering the epitope (containing the precursor sequence of SEQ ID NO: 1); Combined screening: screen high-affinity polypeptides (KD<10⁻ 7 M) by ELISA; ​​​​Blocking validation: competition experiments showed that the candidate peptides (10-50 μg / mL) can dose-dependent block the binding of interfering antibodies to natural tmST2 (inhibition rate >90%), and do not affect the recognition of target antibodies; Specificity confirmation: no cross-reaction, finally determine SEQ ID NO: 1 (GFSPPNISVQWYKNGKEVTLSHESVTQEDSVYTC) as the optimal blocking agent.

[0061] Experimental Example 6 This experimental example is the screening experiment of the first monoclonal antibody and the second monoclonal antibody: Test four candidate antibodies (Ab1, Ab2, Ab3, Ab4) by matrix method; Ab3: against mouse ST2L, no cross-reaction with rat, human ST2L (from R&D Systems, rat-derived monoclonal antibody, clone code DJ8); Ab4: against the unique sequence of sST2 cleavage site (from MD Bioproducts rat anti-human sST2 monoclonal antibody, clone code 10102); Select the antibody pair with the strongest signal and the best specificity, and the antibody combination is shown in Table 7: Table 7

[0062] According to the T-line fluorescence intensity (FI)>2000 (strong signal), and the T-line FI of ST2L extracellular segment cross-reaction group <20 (<1%), and the C-line normal color development for screening. Test Ab1 / Ab2 combination by matrix method, select the antibody pair with the strongest signal and the best specificity.

[0063] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

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.

2. The kit for rapid detection of sST2 according to claim 1, characterized in that, 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.

3. The kit for rapid detection of sST2 according to claim 2, 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%.

4. 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.

5. The kit for rapid detection of sST2 according to claim 4, 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.

6. 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.

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

8. The kit for rapid detection of sST2 according to claim 6, characterized in that, The sealing agent is a BSA solution with a concentration of 5% to 8%.

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