Soluble growth stimulation expression gene 2 protein determination kit, preparation method and application

Abstract

The invention provides a soluble growth stimulation expression gene 2 protein determination kit, a preparation method and application, and belongs to the technical field of biochemical immunoassay. The latex enhanced immunoturbidimetry is adopted to detect the soluble growth stimulation expression gene 2 protein, key components of the reagent 1 and the reagent 2 are optimized and adjusted, preferred components and concentrations of polyanions, a surfactant, an accelerant, a biochemical cross-linking agent and the like are obtained, and the problems that sST2 related reagents are poor in stability and poor in stability are solved. In order to solve the technical problems that in the prior art, the detection accuracy of the high value of sST2 larger than 400 ng / mL cannot be met and the like, the detection accuracy of the high value can be remarkably improved by the optimized components and concentration in the linear range of 5 ng / mL to 432 ng / mL, particularly, the sensitivity, the anti-interference capability, the stability, the repeatability and the front zone of the correspondingly prepared sST2 latex reagent reach and are superior to those of a third-party reagent, and the detection accuracy of the high value of sST2 larger than 400 ng / mL can be remarkably improved by the optimized components and concentration in the linear range of 5 ng / mL to 432 ng / mL. And the requirements of clinical use can be well met.

Description

Technical Field

[0001] This invention relates to the field of biochemical and immunoassay techniques, specifically to a soluble growth-stimulating gene 2 protein assay kit, and further to a method for preparing the soluble growth-stimulating gene 2 protein assay kit and its application in biochemical and immunoassay techniques. Background Art

[0002] The stimulating expression gene 2 (ST2) is located on human chromosome 2 and encodes the ST2 protein (approximately 40 kb). As a member of the interleukin-1 receptor family, it directly influences the progression of heart disease in two main isoforms: transmembrane (ST2L) and soluble (sST2). These two isoforms are formed through alternative splicing and both specifically bind to the functional ligand IL-33. ST2L contains three extracellular immunoglobulin-like domains, one transmembrane domain, and a short cytoplasmic tail. sST2 is the soluble form of ST2L, lacking the transmembrane domain and cytoplasmic tail, and contains a unique 9-amino acid C-terminal sequence. This structure is prone to conformational changes due to environmental factors, resulting in poor stability during in vitro storage. When the body is stimulated by inflammation or tissue damage, related cells induce the expression of sST2. As one of the important biomarkers of the heart, natriuretic peptides only reflect hemodynamic status, while sST2 reflects fibrosis and remodeling. Moreover, sST2 concentration is not affected by factors such as age, sex, race, BMI index, and renal function, and has high specificity. It is closely related to the progression of heart disease. For example, under normal circumstances, sST2 concentration is low (the concentration value is less than 35.0 ng / mL when the immunofluorescence dry quantitative method is used for detection). If heart failure occurs, sST2 concentration increases significantly, and its detection value can be as high as 400 ng / mL or more.

[0003] Currently, common clinical or laboratory methods for detecting the biomarker soluble growth-stimulating gene 2 (sST2) protein include colloidal gold immunochromatography, fluorescence immunochromatography, chemiluminescence immunoassay, and enzyme-linked immunosorbent assay (ELISA). While latex immunoturbidimetry offers high sensitivity, short detection time, and ease of operation, it is less commonly used. Latex-enhanced immunoturbidimetry is a more widely used and more sensitive method. Its basic principle is as follows: specific antibodies are immobilized and coated onto latex particles. When these specific antibodies react with specific antigens in the sample, they aggregate to form larger complex particles. By measuring the significantly amplified turbidity value of these larger particles at a specific wavelength, and since the turbidity value is linearly correlated with the amount of antigen and antibody, a standard curve can be plotted to more sensitively calculate the quantitative concentration of the target substance (such as sST2 protein) in the sample. Immunoassay for sST2 protein detection using latex-enhanced immunoturbidimetry currently faces numerous interfering factors. For example, common substances in the sample, such as rheumatoid factor, triglycerides, and hemoglobin, can easily bind nonspecifically to latex reagents, leading to false positives or false negatives. Furthermore, common technical problems in sST2 protein immunoassay include narrow linear range, low accuracy at high concentrations, and poor kit stability. For instance, while the magnetic microparticle-enzyme-catalyzed chemiluminescence assay (publication number CN110208549A) has expanded the detection range to 1-300 ng / mL, it still cannot meet the detection requirements for high concentrations of sST2 above 400 ng / mL. Although the immunochromatographic test strip invention patent (CN118311272) has been developed... A) The disclosed linear range is 1.00-460.80 ng / mL, but the accuracy of the average of three high-value detection results (407.74) compared to the labeled concentration of the reference standard (460.80 ng / mL) is only 88.49%, indicating that the accuracy of high-value detection remains low. Therefore, this invention addresses these issues by providing a kit, preparation method, and application of a latex-enhanced immunoturbidimetric assay for the determination of sST2 with a wider detection range, higher sensitivity, higher stability, higher accuracy, and higher reliability. Summary of the Invention

[0004] To overcome the above problems, this invention provides a latex-enhanced immunoturbidimetric assay kit for determining soluble growth-stimulating gene 2 protein, which significantly improves performance indicators such as linear range, sensitivity, anti-interference ability, stability, repeatability, and accuracy. The invention also includes the preparation method and application of the kit.

[0005] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a reagent composition for determining soluble growth-stimulating gene 2 protein using a latex-enhanced immunoturbidimetric assay, comprising two independent reagents, reagent 1 and reagent 2; reagent 1 has a pH of 6.0–7.5 and contains a first buffer, an inorganic salt, a first stabilizer, a polyanion, a preservative, a first surfactant, and an accelerator; reagent 2 has a pH of 6.0–7.8 and contains a second buffer, an inorganic salt, a second stabilizer, a preservative, a second surfactant, a biochemical cross-linking agent, and soluble growth-stimulating gene 2 protein antibody-coated latex particles; the final concentration of the inorganic salt in both reagent 1 and reagent 2 is 0.1%–2%; wherein, in reagent 1, the polyanion is selected from 0.3–0.9 g / L. The reagent consists of any one of polyanionic cellulose, polymaleic acid, APAM, and chondroitin sulfate at a concentration of mmol / L; the accelerator is selected from any one or two of PEG6000 and PVP at a concentration of 0.01%–0.3%; in reagent 2, the concentration of soluble growth-stimulating gene 2 protein antibody-coated latex particles is 0.5–2.0 mg / mL; the latex particles used in preparing the soluble growth-stimulating gene 2 protein antibody-coated latex particles are selected from dual-size latex particles, including small-size latex particles and large-size latex particles; the particle size range of the small-size latex particles is 50–150 nm, and the particle size range of the large-size latex particles is 200–420 nm; the preparation of the soluble growth-stimulating gene 2 protein antibody-coated latex particles includes the use of organic compounds as quenchers.

[0006] According to a preferred embodiment of the present invention, in polyanionic polyacrylamide, APAM is the Chinese name for polyacrylamide and the full English name for polyacrylamide.

[0007] According to a preferred embodiment of the present invention, in the accelerator, PEG6000 is called polyethylene glycol 6000 in Chinese and in English.

[0008] According to a preferred embodiment of the present invention, the biochemical crosslinking agent in the accelerator is preferably BS3, which is called bis(sulfosuccinimidyl) suberate sodium salt.

[0009] According to a preferred embodiment of the present invention, in the accelerator, PVP is the Chinese name for polyvinylpyrrolidone, and its full English name is Polyvinyl Pyrrolidone.

[0010] According to a preferred embodiment of the present invention, in the reagent composition, the first buffer and the second buffer are both selected from any one or two of 15-280 mmol / L Tris-HCl buffer, phosphate buffer, HEPES buffer, and MES buffer, and the main components of the first buffer and the second buffer are different; the inorganic salt is selected from any one or two of calcium chloride, sodium chloride, and magnesium chloride; the first stabilizer and the second stabilizer are selected from any one or two of 0.1%-0.5% bovine serum albumin, mannitol, gelatin compounds, casein, and CE series; the preservative is selected from any one or two of sodium azide, benzoic acid, ProClin-300, and antibiotic series; and the preservative... The final concentration of the agent is 0.1% to 3%; the first surfactant is selected from any one or two of alkyl glycosides, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipin, sophorolipid, and A90, at a concentration of 0.05% to 0.2%; the second surfactant is selected from any one or two of alkyl glycosides, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipin, sophorolipid, A90, and P-40, at a concentration of 0.05% to 0.2%; the particle size range of small-diameter latex particles is selected from 50-150 nm, and the particle size range of large-diameter latex particles is selected from 200-420 nm.

[0011] Preferably, in the first and second buffer solutions, the Chinese name of the HEPES buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and the full English name of HEPES is 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid.

[0012] Further preferably, the HEPES buffer has a pH adjustment buffering capacity of 6.0-7.5 and a concentration of 15-150 mg.

[0013] Preferably, in the first buffer and the second buffer, the Chinese name of the MES buffer is 2-(N-morpholino)ethanesulfonic acid buffer, and the full English name of MES is 2-(N-Morpholino)ethanesulfonic acid.

[0014] Further preferably, the MES buffer has a pH adjustment capability of 6.0-7.8 and a concentration of 15-150 mg / L.

[0015] Preferably, in the first stabilizer and the second stabilizer, BSA is the Chinese name for bovine serum albumin, and the full English name for BSA is Bovine Serum Albumin.

[0016] Preferably, in the first stabilizer and the second stabilizer, the Chinese name of the CE series is cellulose ether series, and the preferred representative is ethyl cellulose (EC).

[0017] Preferably, the surfactant includes the first surfactant in reagent 1 and the second surfactant in reagent 2; wherein, a preferred representative of the Triton series is Triton X-100, also known as Triton X-100, or polyethylene glycol octylphenyl ether, or 2-(2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy)ethanol, 2-(2-[4-(1,1,3,3-Tetramethylbutyl)phenoxy]ethoxy)ethanol, or p-iso-octylphenoxy polyethoxyethanol; the chemical name of SDS is sodium dodecyl sulfate, and its full English name is Sodium Dodecyl Sulfate; the chemical name of A90 is polyoxyethylene biphenylene styrenated phenyl ether, and its full product name is EMULGENA-90; the chemical name of B66 is polyoxyethylene tribenzyl phenyl ether, and its full English name is Polyoxyethylene tribenzyl phenyl ether, whose full product name is EMULGEN B-66; P-40 is a nonionic surfactant, whose chemical name is nonylphenyl-polyethylene glycol.

[0018] Further preferably, the surfactant is an alkyl glycoside.

[0019] Preferably, the inorganic salt in reagent 1 is sodium chloride and / or calcium chloride.

[0020] Further preferred inorganic salt is sodium chloride; as an inorganic salt, sodium chloride is relatively more conducive to preventing interference with the binding of enzymes and substrates, and also helps to reduce the viscosity of polyanionic compounds.

[0021] Preferably, the preservative in reagents 1 and 2 is sodium azide.

[0022] Preferably, the first buffer is HEPES or MES buffer; the second buffer is phosphate or MES buffer; the inorganic salt is sodium chloride or calcium chloride; the first stabilizer and the second stabilizer are bovine serum albumin and casein; and the polyanionic stabilizer is APAM or polymaleic acid.

[0023] More preferably, the first buffer is 15~150 mmHEPES buffer and MES buffer.

[0024] More preferably, the second buffer is a 15-150 mm MES buffer and a phosphate buffer.

[0025] More preferably, the pH value of reagent 1 is 6.5.

[0026] More preferably, the pH value of reagent 2 is 6.5.

[0027] More preferably, the inorganic salt is sodium chloride.

[0028] More preferably, the first stabilizer and the second stabilizer are bovine serum albumin.

[0029] More preferably, the preservative is 0.1-3.0% sodium azide.

[0030] More preferably, the first surfactant and the second surfactant are alkyl glycosides.

[0031] More preferably, in reagent 1, the first buffer is HEPES buffer; the first stabilizer is bovine serum albumin; the polyanionic surfactant is APAM; the first surfactant is alkyl glycoside; and the promoter is PVP.

[0032] More preferably, in reagent 2, the second buffer is MES buffer; the second stabilizer is bovine serum albumin; the second surfactant is alkyl glycoside; the concentration of soluble growth-stimulating gene 2 protein antibody-coated latex particles in reagent 2 is 0.75–1.75 mg / mL, and the particle size range of the large-diameter latex particles is selected from 300–420 nm.

[0033] Secondly, the present invention provides a soluble growth-stimulating gene 2 protein assay kit, comprising the above-mentioned reagent composition, and further comprising quality control and calibrators, wherein the quality control and calibrators each contain at least two concentration levels of anti-soluble growth-stimulating gene 2 recombinant protein, and the buffers are all MES buffers containing human serum.

[0034] According to a preferred embodiment of the present invention, in the soluble growth-stimulating gene 2 protein assay kit, the quality control comprises at least two concentration levels of soluble growth-stimulating gene 2 recombinant protein, namely: Level 1: 20 ng / mL to 60 ng / mL; Level 2: 80 ng / mL to 130 ng / mL; the calibrator comprises at least six concentration levels of soluble growth-stimulating gene 2 recombinant protein, namely: 0 ng / mL, 10 ng / mL, 54 ng / mL, 108 ng / mL, 216 ng / mL and 432 ng / mL.

[0035] Thirdly, the present invention provides a method for preparing latex particles coated with a soluble growth-stimulating gene 2 protein antibody, comprising the following steps: S1: taking dual-size latex particles and suspending them in 10-20... S1: In mM MES buffer, adjust the concentration of dual-size latex particles to 30-70 mg / L; S2: Add EDC and NHS, and react at room temperature for 1 hour to catalyze the formation of amide bonds between the carboxyl groups on the surface of the latex particles and the amino groups on the antibody; S3: Quench the EDC with 0.05-0.2M quencher for no more than 10 minutes to terminate the catalytic activity of unreacted EDC; S4: Add an equimolar amount of human soluble growth-stimulating gene 2 protein antibody to the dual-size latex particles, and block the reaction at room temperature for 1.5-3 hours to obtain a crude solution containing latex particles coated with soluble growth-stimulating gene 2 protein antibody; S5: Remove the supernatant from the crude solution containing soluble growth-stimulating gene 2 protein antibody coated with latex particles by low-temperature high-speed centrifugation or filtration to obtain the stock solution of soluble growth-stimulating gene 2 protein antibody coated with latex particles; S6: Reuse 10-20 Dilute the soluble growth-stimulating gene 2 protein antibody-coated latex particles to 10 g / L with mM MEM buffer, adjust the pH to 6.0-7.8 to obtain the diluted solution of the soluble growth-stimulating gene 2 protein antibody-coated latex particles; aliquot and store at 4℃ for later use.

[0036] According to a preferred embodiment of the present invention, in step S1 of the method for preparing soluble growth-stimulating gene 2 protein antibody-coated latex particles, the pH value of the 10-20 mM MES buffer is 5.

[0037] According to a preferred embodiment of the present invention, in the method for preparing soluble growth-stimulating gene 2 protein antibody-coated latex particles, in step S1: the particle size of the small-diameter latex particles is 50-100 nm, the particle size of the large-diameter latex particles is 300-420 nm, and the final concentration after equimolar mixing of the small-diameter and large-diameter latex particles is 30-70 mg / L; in step S2: the concentration of EDC is 12-15 mM, and the concentration of NHS is 35-50 mM; in step S3: the quencher is an organic compound, either QSY-21 or TCEP.

[0038] Fourthly, the present invention provides a method for preparing a soluble growth-stimulating gene 2 protein assay kit, used to prepare the above-mentioned reagent composition, wherein the reagent 1 preparation process is as follows: take the first buffer solution and add it to the container, and heat it at 300-400... The mixture is stirred at a speed of rpm. Then, the inorganic salt, first stabilizer, polyanionic agent, preservative, first surfactant, and accelerator from reagent 1 are added sequentially to the first buffer solution according to the predetermined components and concentrations, and mixed thoroughly. Finally, the pH of reagent 1 is adjusted to 6.0–7.5 using a pH adjuster, thus obtaining reagent 1. The preparation process for reagent 2 is as follows: First, the second buffer solution is added to a container. The inorganic salt, second stabilizer, preservative, and second surfactant are added sequentially to the second buffer solution according to the predetermined components and concentrations, and mixed thoroughly. The pH is adjusted to 6.0–7.8, completing the preparation of the reagent 2 stock solution. The reagent 2 stock solution is added to the dilution solution of the soluble growth-stimulating gene 2 protein antibody-coated latex particles prepared above, further diluting the soluble growth-stimulating gene 2 protein antibody-coated latex particles to a final concentration of 0.5–2.0 mg / mL, and mixed thoroughly. Finally, the pH is adjusted to the range of 6.0–7.8, thus obtaining reagent 2.

[0039] According to a preferred embodiment of the present invention, in the preparation method of the soluble growth-stimulating gene 2 protein assay kit, the final concentration of the soluble growth-stimulating gene 2 protein antibody-coated latex particles is 0.75-1.75 mg / mL.

[0040] Fifthly, the present invention also claims protection for the application of the above-mentioned reagent composition, the soluble growth-stimulating gene 2 protein assay kit, the method for preparing soluble growth-stimulating gene 2 protein antibody-coated latex particles, and the method for preparing the soluble growth-stimulating gene 2 protein assay kit in the preparation of latex reagent-related products.

[0041] The beneficial effects of the present invention are: This invention provides a detection method, an immunoassay kit, preparation method, and application of soluble growth-stimulating gene 2 protein using latex-enhanced immunoturbidimetric assay. The invention detects the sST2 content in serum samples by coating human sST2 antibody onto dual-size latex particles. When sST2 in the serum sample undergoes a specific immune reaction with the soluble growth-stimulating gene 2 protein antibody-coated latex particles (hereinafter referred to as sST2 antibody-coated latex particles), aggregates are formed. By measuring the turbidity of the aggregates and corresponding to the standard curve of the standard, the content of the analyte in the sample can be determined.

[0042] This invention mainly uses latex-enhanced immunoturbidimetry for detection, and optimizes the components, concentrations, and preparation processes in reagents 1 and 2. For example, polyanionic polyacrylamide (APAM) is preferred in reagent 1, and an additional accelerator is added. Alkyl glycoside is preferred as the surfactant, and polyvinylpyrrolidone (PVP) is preferred as the accelerator. By selecting the pairing of latex particles with different particle sizes, the optimal buffer solution, stabilizer and accelerator, and the optimal pH value of the reagents, the ability to resist interference from chylomicron particles is significantly improved.

[0043] In the optimization process of the sST2 latex reagent provided by this invention, regarding the buffer solution, the first buffer solution is preferably HEPES buffer. Because HEPES buffer relies on the protonation / deprotonation reaction of the two nitrogen atoms on the piperazine ring and the acid-base balance ability of the sulfonic acid group (R-SO3H), it can effectively resist external pH changes, making the reagent more stable. Regarding stabilizers, it is generally believed that ionic stabilizers (such as SDS) can effectively stabilize latex at a concentration of 0.1-1%, but concentrations exceeding 2% may cause flocculation; while non-ionic stabilizers (such as bovine serum albumin BSA) can maintain latex stability at a concentration of 0.5-2%, but a concentration of 5% may cause particle aggregation. However, preliminary experiments of this invention have found that, regardless of whether the stabilizer is ionic or non-ionic, a concentration exceeding 0.5% is not conducive to maintaining latex stability. Therefore, a low concentration of non-ionic stabilizer in the range of 0.1-0.5%, such as bovine serum albumin, is the preferred stabilizer. Regarding the polyanion, the preferred component of this invention is APAM. It is speculated that APAM can promote a specific immune response between sST2 antibody-coated latex particles and sST2 antigen in the sample, accelerating particle aggregation and inhibiting the increase in signal-to-noise ratio and the occurrence of non-specific reactions. Regarding the promoter, this invention preferably uses a low concentration range of 0.01~0.3% PVP. This is likely because in serum samples, low concentrations of PVP can shield the charge neutralization effect of electrolytes or proteins on latex, preventing false aggregation. Furthermore, the carbonyl group on the PVP molecule can form hydrogen bonds with the carboxyl group of the antibody, assisting the antibody in specifically binding to the surface of sST2 antibody-coated latex particles, improving the specific antibody-antigen binding efficiency (increasing the specific aggregation rate between latex particles, dual-size antibodies, and sST2 antigen). It also ensures that the turbidity change is linearly related to the concentration of sST2 antigen in the sample. The direct effect is to improve the detection sensitivity of this invention, expand the linear detection range, and improve the accuracy of high-value detection. It also provides a positive effect in terms of anti-interference capability. Regarding the biochemical cross-linking agent, the addition of bis(succinimide) octanoate sodium salt (BS3) to reagent 2 can form amide bonds with the protein primary amine through the reaction of its NHS ester group, making the reagent performance more stable. More importantly, this invention can still maintain linearity at high magnification front bands (such as 2-3 times higher values), that is, the detection line for high concentrations is significantly improved by 2 and 3 times. It can be seen that the sST2 latex reagent with the preferred components effectively avoids the front band effect, can measure higher concentration samples, further broaden the detection range, and improve the detection upper limit.Conventional latex reagents are affected by the pre-band effect, requiring pre-dilution of high-concentration samples that exceed the linear range. This may introduce errors and increase operation time. However, this invention, by introducing BS3, can overcome the limitations of the pre-band effect and directly detect high-magnification pre-band samples. This not only reduces sample dilution steps and human error but also improves detection efficiency. It is particularly suitable for accurate monitoring of diseases such as inflammation or heart failure with abnormally elevated sST2 concentrations, and can detect abnormal changes in sST2 concentrations more promptly.

[0044] Regarding inorganic salts, the purpose is to adjust the ionic strength of the reagents to ensure the reaction system is in a suitable osmotic pressure environment, preventing latex particles from self-aggregating or antibodies from detaching due to osmotic pressure imbalance. Sodium chloride or calcium chloride is preferred, and the inorganic salt must contain at least sodium chloride. Sodium chloride effectively avoids interference from the specific binding reaction between sST2 and sST2 antibody-coated latex particles in the test sample, and it also helps reduce the viscosity of polyanionic compounds. During the screening process, it was also found that the presence of calcium chloride in the inorganic salt helps resolve the hook effect, presumably due to the presence of calcium chloride. 2+ By neutralizing the charge, the antigen-antibody binding is preferentially promoted, which to some extent helps to solve the problem of low detection values ​​for high concentrations of sST2. In reagents 1 and 2, the optimal concentration of the inorganic salt depends on the nature of the analyte, the surface characteristics of the latex particles, the requirements of the reaction system, and the properties of the inorganic salt itself. If the analyte is a small molecule antigen or antibody, or if rapid aggregation of latex particles is required, and the inorganic salt is compatible with other components of the reaction system, the concentration of the inorganic salt can generally be appropriately increased. For example, in patent (CN119395307A), when the analyte is SAA, it was found that high concentrations of inorganic salts (such as NaCl) can shield the charge on the surface of latex particles, thereby promoting particle aggregation and improving detection sensitivity. However, the effect of the inorganic salt concentration on detection performance needs to be verified experimentally. Due to the complexity and diversity of components in latex reagents, blindly applying empirical values ​​for common inorganic salt concentrations is ineffective. In this invention, due to the instability of antibodies against soluble growth-stimulating gene 2 (sST2) protein and the inherent technical problems of high-value detection accuracy, this invention adopts a technical solution of coating two-size latex particles with human sST2 antibodies. In order to ensure that the stability between the two-size latex particles is not affected, the inorganic salt concentration screening experiment found that a low concentration range of 0.1-2% is beneficial to maintaining the stability of the latex reagent. Even when the inorganic salt concentration in reagent 1 is as low as 0.01%, the stability of the reagent can still meet the requirements. However, if the inorganic salt in reagent 2 is high (4%), the fluctuation of the detection values ​​in Example 26 and Comparative Example 4 cannot meet the stability requirements. That is, high concentration of inorganic salt is not conducive to maintaining the stability of sST2 latex reagent.

[0045] The present invention optimizes the preparation process of sST2 latex reagent, particularly the preparation process of sST2 antibody-coated latex particles. By selecting dual-size latex particles and adding a quencher, the sensitivity and linear range of the prepared sST2 latex reagent are significantly improved. This also enhances key performance indicators such as the reagent's anti-interference ability, stability, and repeatability. Specifically, the dual-size latex particles are carboxyl microspheres of two different sizes. The smaller-size latex microspheres (50-150 nm) have a higher number of carboxyl groups and a larger specific surface area, allowing them to bind a large amount of antibody, thus improving the reagent's linear range. The larger-size latex microspheres (200-420 nm) can capture low concentrations of antibody, enhancing low-value precision. The use of organic compounds as quenchers during the latex particle coating process further enhances the performance of the sST2 latex reagent. The quenchers terminate the catalytic activity of unreacted activators (EDCs), preventing unbound activators from continuing to react non-specifically with the latex particle surface or subsequently added antibodies / antigens. This improves the specificity and stability of the sST2 antibody-coated latex particles for detection. Simultaneously, the quenching reaction helps ensure that the antibody specifically binds to the latex particle surface without interference from unreacted activators, thus optimizing the coating effect. In particular, the quenchers are organic compounds, possessing advantages such as high stability, good compatibility, and low toxicity. This invention preferably uses QSY-21 as the quencher. Experimental results show that adding quenchers is beneficial for improving the sensitivity, linear range, and stability of the sST2 latex reagent.

[0046] Latex reagents contain numerous components, and the preferred combination of these components in this invention has a positive synergistic effect on improving the overall performance of the reagent. For example, regarding sensitivity, the use of quenchers, the type of quencher, and the combination of dual-size latex particles during the preparation of the sST2 antibody-coated latex particles have a synergistic and positive impact on improving the sensitivity of the sST2 latex reagent. Regarding linear range, the pairing of dual-size latex particles, and the preferred components and concentrations of added polyanions, accelerators, and biochemical cross-linking agents have a positive impact on expanding the linear range of detection and improving the accuracy of high-value (3 times that of 432 ng / mL) detection. Regarding interference resistance, the preferred conditions for dual-size latex particle pairing, buffer solution, stabilizer, accelerator, and pH value also have a synergistic and positive impact on improving the reagent's resistance to interference. Furthermore, the preferred conditions for buffer solution, stabilizer, polyanions, and surfactants also have a synergistic and positive impact on improving the stability of the reagent.

[0047] The performance indicators of the sST2 latex reagent provided by this invention have achieved significant improvements overall. This invention optimizes the five main components in reagents 1 and 2: buffer solution, inorganic salts, stabilizers, surfactants, and preservatives. Furthermore, it creatively incorporates polyanionic polymers, accelerators, and biochemical cross-linking agents into reagent 1. In reagent 2, the preparation process of coating latex particles with soluble growth-stimulating gene 2 protein antibodies is optimized through the pairing and combination of dual-size latex particles, the introduction of a quenching process, and the addition of biochemical cross-linking agents. This improves the stability of the chemical coupling between the dual-size latex particles and the soluble growth-stimulating gene 2 protein antibodies, further enhancing the sensitivity, linear range, and anti-interference ability for detecting sST2 protein. In addition, the sST2 latex reagent prepared by this invention exhibits superior performance in terms of stability and repeatability. Specific experiments also verify a high correlation between the preferred embodiment 11 of this invention and the detection results of third-party reagents. This invention not only provides a specific sST2 latex reagent, but also constructs a more optimized detection platform for sST2 protein determination using latex-enhanced immunoturbidimetric assay, and a method for preparing a kit for detecting soluble growth-stimulating gene 2 protein using latex-enhanced immunoturbidimetric assay. Through comprehensive performance evaluation and experimental verification with specific clinical samples, the detection sensitivity, specificity, linear range, anti-interference ability, stability, repeatability, and accuracy for detecting high-value sST2 protein have all been significantly improved. The coefficient of variation (CV%) of the sensitivity detection value can reach 0.13%, and the linear detection range can reach 5 ng / mL to 432 ng / mL. The concentration of sST2 latex reagent against rheumatoid factor (RF) interference can reach up to 600 mmol / L, against hemoglobin (HB) interference up to 800 mmol / L, and against triglyceride (TG) interference up to 1.5%. The reagent is stable for 24 months at 2-8℃, with minimal fluctuations in detection values ​​after long-term low-temperature storage. The coefficient of variation between 10 repeated measurements is as high as 0.7%. These performance indicators meet or exceed the level of third-party reagents (high-sensitivity magnetic particle chemiluminescence method). The agreement rate between the measured values ​​of preferred Example 11 and the third-party reagent remains good (R...). 2 = 0.9938), meaning that the detection results of the sST2 latex reagent provided by this invention are highly linearly correlated with the gold standard (chemiluminescence), making it suitable for preliminary screening in clinical auxiliary testing. It is evident that the sST2 latex reagent provided by this invention not only possesses the advantages of latex reagents—simple operation, low cost, good stability, rapid detection, and good repeatability—but also achieves the high sensitivity and wide linear range characteristic of magnetic particle chemiluminescence.

[0048] This invention provides a soluble growth-stimulating gene 2 (sST2) protein assay kit, its preparation method, and its application. It optimizes the preparation process of key components such as buffer, inorganic salts, stabilizers, surfactants, and preservatives in reagents 1 and 2, as well as the preparation process of soluble sST2 protein antibody-coated latex particles. Through optimized preparation of the soluble sST2 protein assay kit, and verification using specific experiments and clinical samples, this invention demonstrates that the sST2 latex reagent significantly improves the overall performance of latex-enhanced immunoturbidimetric assay for determining soluble sST2 protein. In particular, it addresses the technical problems of a narrow linear range for sST2 detection and the inability to achieve high-concentration sST2 values. Overall, it improves the sensitivity, anti-interference ability, stability, repeatability, and accuracy of the sST2 latex reagent. Combined with a fully automated biochemical analyzer, it enables automated detection and is particularly suitable for monitoring or evaluating the severity of heart failure at high sST2 concentrations in clinical settings. Attached Figure Description

[0049] Figure 1 The graph shows the linear evaluation results of the sST2 latex reagent prepared in Example 11 of this invention and the third-party reagent. The horizontal axis represents the theoretical value and the vertical axis represents the measured value. The unit is ng / mL. S11 represents Example 11 and A represents the third-party reagent. Figure 2 The graph shows the stability evaluation results of the sST2 latex reagent and third-party reagent prepared in 37 examples and 6 comparative examples of this invention. In the graph, A represents the third-party reagent, S1-S37 represent the 37 examples, and D1-D6 represent the 6 comparative examples. The horizontal axis represents the storage time at 2-8℃, with a whole month as the unit of measurement, and the vertical axis represents the quantitatively determined concentration value, with the unit being ng / mL. Figure 3 This is a graph showing the correlation evaluation results between the measured values ​​of the sST2 latex reagent prepared in Example 11 of this invention and the measured values ​​of the third-party reagent. The horizontal axis represents the measured values ​​of Example 11, in ng / mL, and the vertical axis represents the measured values ​​of the third-party reagent, in ng / mL. Detailed Implementation

[0050] Exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein the same or similar terms represent the same concepts, such as soluble growth-stimulating gene 2 protein = sST2 protein = sST2. In the following detailed description, numerous specific details are set forth to provide a full understanding of the embodiments disclosed herein for ease of explanation. The following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Specifically, the soluble growth-stimulating gene 2 recombinant protein (catalog number: LA446) was purchased from Medix Biochemica; the anti-human soluble growth-stimulating gene 2 protein antibody (human soluble growth-stimulating gene 2 protein antibody, abbreviated as human sST2 antibody, catalog number: 100686) was purchased from Medix Biochemica; A90 and B66 were purchased from Kao Corporation's EMULGEN series of nonionic surfactants (EMULGEN A-90 and EMULGEN). B-66); QSY-21 (carboxylic acid succinimide ester, belonging to amine reactive quenchers, catalog number: Q20132) was purchased from Thermo Fisher Scientific; alkyl glycoside (catalog number: A864768) was purchased from Macklin Scientific; latex particles were purchased from JH&T (Beijing) Biotechnology Co., Ltd.; activators EDC (catalog number: N808856) and NHS (catalog number: N811124) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; BS3 (bissuccinimide octanoate sodium salt, catalog number: CLS-HT0195) was purchased from Shanghai Chuangsai Technology Co., Ltd.; the fully automated biochemical analyzer was purchased from Hitachi, Japan, model 7180; the third-party reagent was the sST2 assay kit (magnetic microparticle chemiluminescence method) purchased from Beijing Leadman Biochemical Co., Ltd. However, it is obvious that one or more examples can be implemented without these specific details, and conditions not specifically specified in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified in the following examples, the molecular biology experimental methods were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0052] The application objects measured in this invention refer to biological samples of bodily fluids collected from human or animal bodies. After the sample collection is completed, the biological sample has been separated from the living human or animal body. Examples include blood samples (whole blood / serum / plasma), bodily fluids, tissues, excrement, and isolated cultures (blood cultures, sputum cultures, etc.), all of which are inanimate biological samples outside the body. The measurement process is completed in vitro. The direct purpose of the measurement is to determine whether the target protein (soluble growth-stimulating gene 2 protein) or its amino acids derived from the degradation products of the target protein is present in the sample. The measurement results help doctors interpret the information obtained from medical history. This measurement belongs to the determination of components or contents in inanimate biological samples in vitro. There is no process of directly obtaining a disease diagnosis result or health status during the measurement. Therefore, this invention does not belong to the diagnostic method for diseases and meets the basic requirements of the Patent Law for the subject matter of patent protection. The specific embodiments of this invention are described below.

[0053] This invention optimizes the composition, concentration, and preparation process of reagents 1 and 2, which are independent of each other, in the determination of soluble growth-stimulating gene 2 (sST2) protein using a latex-enhanced immunoturbidimetric assay. Regarding the key components, reagent 1 includes a first buffer, inorganic salts, a first stabilizer, a polyanion, a preservative, a first surfactant, and an accelerator; reagent 2 includes a second buffer, inorganic salts, a second stabilizer, a preservative, a second surfactant, and soluble growth-stimulating gene 2 protein antibody-coated latex particles (referred to as sST2 antibody-coated latex particles).

[0054] The first and second buffer solutions are preferably any one or two of Tris-HCl buffer, phosphate buffer, MES buffer, and HEPES buffer at a concentration of 50–150 mmol / L, and preferably the main components of the first and second buffer solutions are different; the inorganic salts are preferably any one or two of calcium chloride, sodium chloride, and magnesium chloride, and the preferred concentration of the inorganic salts in reagents 1 and 2 is 0.1%–2%; the first and second stabilizers are preferably any one or two of bovine serum albumin, mannitol, gelatin compounds, casein, and CE series at a concentration of 0.1%–0.5%; the preservatives are preferably any one or two of sodium azide, benzoic acid, ProClin-300, and antibiotic series at a concentration of 0.1%–3%; the first and second surfactants are preferably alkyl glycosides, lecithin, Tween 20, and Triton series (preferably Triton) at a concentration of 0.05%–0.2%. Reagent 1 also includes: 0.3–0.9 mmol / L of a polyanionic acid, selected from polyanionic cellulose, polymaleic acid, polyacrylamide (APAM), and chondroitin sulfate; 0.01%–0.3% of an accelerator, selected from polyethylene glycol 6000 (PEG6000) and polyvinylpyrrolidone (PVP); and 0.05%–0.2% of a second surfactant, selected from alkyl glycosides, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipids, sophorolipids, A90, and nonylphenyl polyethylene glycol (P-40).

[0055] Regarding the preparation method, the preparation of the soluble growth-stimulating gene 2 protein assay kit of the present invention includes: first, preparing reagent 1; then, based on the preparation of the stock solution of reagent 2, preparing dual-size latex particles coated with anti-human soluble growth-stimulating gene 2 protein antibody (hereinafter referred to as soluble growth-stimulating gene 2 protein antibody-coated latex particles, or sST2 antibody-coated latex particles); then, preparing reagent 2; and finally, preparing reagent 1, reagent 2, and quality control or standard products into a kit according to the specifications.

[0056] The preparation method of reagent 1 is as follows: take the first buffer solution and add it to the container, stir at a speed of 300-400 rpm, then add the inorganic salt, the first stabilizer, the polyanionic agent, the preservative, the first surfactant and the accelerator to the buffer solution in the order of the predetermined components and their concentrations and mix well. Finally, use a pH adjuster to adjust the pH of reagent 1 to a pH value of 6-7.5, and thus prepare reagent 1.

[0057] The preparation method of reagent 2 stock solution is as follows: take the second buffer solution and add it to the container, stir at a speed of 300-400 rpm, then add the inorganic salt, the second stabilizer, the preservative, the biochemical cross-linking agent and the second surfactant to the second buffer solution in the order of the predetermined components and their concentrations and mix well. Finally, adjust the pH value to 6.0-7.8 with a pH adjuster, which is the reagent 2 stock solution.

[0058] The preparation process of sST2 antibody-coated latex particles is as follows: 1) Take small-diameter (50-100nm or 100-150nm) latex particles and large-diameter (200-300nm or 300-420nm) latex particles in a 1:1 volume ratio, add 10-20mM, pH 5.0 MES buffer and mix well to make the concentration of the two-diameter latex particles 30-70mg / L; 2) Add 12-15 Crosslinking reaction of 1 mM EDC and 35-50 mM NHS at room temperature (20-25℃) for 1 hour; wherein, the chemical name of EDC is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; the chemical name of NHS is N-hydroxysuccinimide; 3) Add 0.1-0.25M quencher (QSY-21 or TCEP) to quench EDC for no more than 10 minutes; 4) Add human sST2 antibody in an equimolar amount to the two-particle-size latex particles, and block the reaction in a shaker at room temperature (20-25℃) for 1.5-3 hours to obtain a crude solution containing sST2 antibody-coated latex particles; 5) Remove the supernatant from the above crude solution by low-temperature high-speed centrifugation or filtration to obtain the stock solution of sST2 antibody-coated latex particles; 6) Dilute the sST2 antibody-coated latex particles again with 10-20 mM, pH 5.0 MEM buffer to 10 The concentration of the solution was adjusted to g / L, and the pH was adjusted to 6.0-7.8 to obtain a diluted solution of sST2 antibody-coated latex particles. During the preparation process, quenching aims to utilize the active ester groups, amino groups, and other organic functional groups on the quencher to react with the active groups of EDC, causing EDC to lose its catalytic ability. In the quencher, QSY-21 is chemically named carboxylic acid succinimide ester, and its full English name is QSY21 carboxylic acid, NHS Ester; TCEP is chemically named tris(2-carboxyethyl)phosphine.

[0059] The preparation method of reagent 2 is as follows: Add the prepared stock solution of reagent 2 to the diluent of sST2 antibody-coated latex particles, further dilute the sST2 antibody-coated latex particles to a final concentration of 0.5-2.0 mg / mL (preferably within the range of 0.75-1.75 mg / mL), mix well, and adjust the pH to the range of 6.5-7.8 to obtain reagent 2.

[0060] In the latex-enhanced immunoturbidimetric assay kit for detecting sST2 (hereinafter referred to as sST2 latex reagent), the preferred volume ratio of reagent 1 to reagent 2 is 4:1. Both the quality control and standard samples are prepared from a liquid matrix containing soluble growth-stimulating gene 2 recombinant protein, either human serum or a serum-like matrix, with the human serum content being no less than 5%. The specific preparation process for the quality control is as follows: soluble growth-stimulating gene 2 recombinant protein antigen is added to MES buffer to prepare two levels of quality control. The target value range for level 1 is 20-60 ng / mL, and the target value range for level 2 is 80-130 ng / mL. The specific preparation process for the standards is as follows: Soluble growth-stimulating gene 2 recombinant protein is added to 15-100 mmol / L LMES buffer and mixed to prepare five concentration series. The target concentrations of soluble growth-stimulating gene 2 recombinant protein are 432 ng / mL, 216 ng / mL, 108 ng / mL, 54 ng / mL, and 10 ng / mL, respectively. In addition, the standard series also includes human serum standards without soluble growth-stimulating gene 2 recombinant protein, i.e., the concentration of soluble growth-stimulating gene 2 recombinant protein is 0 ng / mL. 5-10% human serum is then added to the six concentration series standards (0 ng / mL, 10 ng / mL, 54 ng / mL, 108 ng / mL, 216 ng / mL, and 432 ng / mL).

[0061] This invention, based on the overall experimental design of the latex-enhanced immunoturbidimetric assay kit for detecting sST2, includes 37 sets of examples encompassing the aforementioned core components, concentrations, and preparation methods. The aim is to screen for optimal components and concentrations of different buffers, inorganic salts, stabilizers, preservatives, and surfactants. It also verifies the addition of polyanions and accelerators to Reagent 1, and the use of a combination of latex particles with significantly different particle sizes in Reagent 2, along with the optimization of the sST2 antibody-coated latex particle preparation process by adding quenchers, thereby improving reagent specificity and sensitivity. Furthermore, six comparative examples are included to verify the maximum usable concentrations of buffers, inorganic salts, stabilizers, polyanions, surfactants, and accelerators, particularly to identify the optimal concentration ratio of polyanions and surfactants to enhance the synergistic effect on the performance indicators of the latex-enhanced immunoturbidimetric assay kit for detecting sST2.

[0062] The key components, concentrations, and preparation processes of the latex-enhanced immunoturbidimetric assay kit for detecting sST2 are as follows: The kits prepared using these examples and comparative examples were used to verify their respective linear range, sensitivity, anti-interference ability, stability, and repeatability, and the specific detection results are as follows.

[0063] Example 1: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.4% PEG6000, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0064] Example 2: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.04% PEG6000, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 100 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0065] Example 3: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 150 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0066] Example 4: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 100 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L polymaleic acid, the preservative is 3% sodium azide, the first surfactant is 0.05% B66, the accelerator is 0.01% PEG6000, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0067] Example 5: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 100 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L polymaleic acid, the preservative is 3% sodium azide, the first surfactant is 0.05% B66, the accelerator is 0.4% PVP, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 100 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0068] Example 6: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 100 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L polymaleic acid, the preservative is 3% sodium azide, the first surfactant is 0.05% B66, the accelerator is 0.04% PVP, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 150 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0069] Example 7: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 150 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% A90, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0070] Example 8: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 150 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L polymaleic acid, the preservative is 3% sodium azide, the first surfactant is 0.05% A90, the accelerator is 0.01% PEG6000, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 100 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0071] Example 9: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 150 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% A90, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.0; In reagent 2, the second buffer is 150 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0072] Regarding the preparation of reagent 2 in Examples 1-9, during the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles was 100-150 nm, the particle size of large-diameter latex particles was 300-420 nm, the concentration of MES buffer at pH 5.0 was 10 mM, the concentration of dual-diameter latex particles was 30 mg / L, the crosslinking agent was 15 mM EDC and 50 mM NHS, and the quencher was 0.05 M QSY-21. After the blocking reaction with human sST2 antibody was added for 3 hours, the pH of the dilution of sST2 antibody-coated latex particles was adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 was 0.75 mg / mL, and the pH of reagent 2 was finally adjusted to 6.5.

[0073] Example 10: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic polymer is 0.3 mmol / L APAM, the preservative is 0.3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0074] Example 11: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 2% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, the second surfactant is 0.05% alkyl glycoside, and the biochemical crosslinking agent is 0.01% BS3.

[0075] Example 12: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salts are 2% sodium chloride and calcium chloride (i.e., 1% sodium chloride and 1% calcium chloride), the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, the second surfactant is 0.05% alkyl glycoside, and the biochemical crosslinking agent is 0.01% BS3.

[0076] Example 13: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salts are 1% sodium chloride and calcium chloride (i.e., 0.5% sodium chloride and 0.5% calcium chloride), the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0077] Example 14: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salts are 0.4% sodium chloride and calcium chloride (i.e., 0.2% sodium chloride and 0.2% calcium chloride), the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0078] Example 15: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 2% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L polymaleic acid, the preservative is 0.3% sodium azide, the first surfactant is 0.05% Triton X-100, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salts are 2% sodium chloride and magnesium chloride (i.e., 1% sodium chloride and 1% magnesium chloride), the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, and the second surfactant is 0.05% P-40.

[0079] Example 16: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 2% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic acid is 0.3 mmol / L polymaleic acid, the preservative is 0.3% sodium azide, the first surfactant is 0.05% rhamnolipid, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salts are 1% sodium chloride and magnesium chloride (i.e., 0.5% sodium chloride and 0.5% magnesium chloride), the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, and the second surfactant is 0.05% P-40.

[0080] Example 17: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 2% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L polymaleic acid, the preservative is 0.3% sodium azide, the first surfactant is 0.05% sophorolipid, and the accelerator is 0.01% PEG6000. The pH of reagent 1 is adjusted to 6.5. In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salts are 0.4% sodium chloride and magnesium chloride (i.e., 0.2% sodium chloride and 0.2% magnesium chloride), the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, and the second surfactant is 0.05% P-40.

[0081] Regarding the preparation of reagent 2 in Examples 10-17, during the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles was 50-100 nm, the particle size of large-diameter latex particles was 300-420 nm, the concentration of MES buffer at pH 5.0 was 15 mM, the concentration of dual-diameter latex particles was 40 mg / L, the crosslinking agents were 14 mM EDC and 45 mM NHS, and the quencher was 0.15 M QSY-21. After the blocking reaction with human sST2 antibody was added for 2 hours, the pH of the dilution of sST2 antibody-coated latex particles was adjusted to 6.0. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 was 1.25 mg / mL, and the pH of reagent 2 was finally adjusted to 6.5.

[0082] Example 18: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 200 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% D-mannitol, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% lecithin, and the promoter is 0.01% PEG6000. The pH of reagent 1 is adjusted to 7. In reagent 2, the second buffer is 200 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.3% bovine serum albumin, the preservative is 3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0083] Example 19: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 100 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% gelatin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% Tween 20, the accelerator is 0.01% PEG6000, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is 200 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.5% bovine serum albumin, the preservative is 3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0084] Example 20: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L phosphate buffer, the inorganic salts are 0.1% sodium chloride and magnesium chloride (i.e., 0.05% sodium chloride and 0.05% magnesium chloride), the first stabilizer is 0.3% D-mannitol, the polyanionic cellulose is 0.3 mmol / L polyanionic cellulose, the preservative is 0.1% sodium azide, the first surfactant is 0.05% SDS, the accelerator is 0.01% PEG6000, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is 50 mmol / L phosphate buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% D-mannitol, the preservative is 3% sodium azide, and the second surfactant is 0.05% A90.

[0085] Example 21: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 200 mmol / L Tris-HCl buffer, the inorganic salts are 0.1% sodium chloride and magnesium chloride (i.e., 0.05% sodium chloride and 0.05% magnesium chloride), the first stabilizer is 0.3% gelatin, the polyanionic is 0.3 mmol / L polyanionic cellulose, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is 100 mmol / L Tris-HCl buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.3% D-mannitol, the preservative is 3% sodium azide, and the second surfactant is 0.05% A90.

[0086] Example 22: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 100 mmol / L MEM buffer, the inorganic salts are 0.1% sodium chloride and magnesium chloride (i.e., 0.05% sodium chloride and 0.05% magnesium chloride), the first stabilizer is 0.3% casein, the polyanionic cellulose is 0.3 mmol / L polyanionic cellulose, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is 100 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.5% D-mannitol, the preservative is 3% sodium azide, and the second surfactant is 0.05% A90.

[0087] Example 23: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L Tris-HCl buffer, the inorganic salts are 0.1% sodium chloride and calcium chloride (i.e., 0.05% sodium chloride and 0.05% calcium chloride), the first stabilizer is 0.5% gelatin, the polyanionic surfactant is 0.3 mmol / L chondroitin sulfate, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is 100 mmol / L MEM buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% gelatin, the preservative is 3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0088] Example 24: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is a 200 mmol / L phosphate buffer, the inorganic salts are 0.1% sodium chloride and calcium chloride (i.e., 0.05% sodium chloride and 0.05% calcium chloride), the first stabilizer is 0.5% casein, the polyanionic surfactant is 0.3 mmol / L chondroitin sulfate, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is a 50 mmol / L Tris-HCl buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.3% gelatin, the preservative is 3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0089] Example 25: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 100 mmol / L MEM buffer, the inorganic salts are 0.1% sodium chloride and calcium chloride (i.e., 0.05% sodium chloride and 0.05% calcium chloride), the first stabilizer is 0.5% bovine serum albumin, the polyanionic surfactant is 0.3 mmol / L chondroitin sulfate, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is 50 mmol / L phosphate buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.5% gelatin, the preservative is 3% sodium azide, and the second surfactant is 0.05% alkyl glycoside.

[0090] Regarding the preparation of reagent 2 in Examples 18-25, during the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles was 50-100 nm, the particle size of large-diameter latex particles was 200-300 nm, the concentration of MES buffer at pH 5.0 was 15 mM, the concentration of dual-diameter latex particles was 50 mg / L, the crosslinking agents were 13 mM EDC and 40 mM NHS, and the quencher was 0.1 M QSY-21. After the human sST2 antibody was added and the blocking reaction was carried out for 1.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles was adjusted to 6.0. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 was 1.75 mg / mL, and the pH of reagent 2 was finally adjusted to 6.5.

[0091] Example 26: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% D-mannitol, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 4% sodium chloride, the second stabilizer is 0.1% casein, the preservative is 2% sodium azide, and the second surfactant is 0.1% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction of human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 2 mg / mL, and the pH of reagent 2 is accurately adjusted to 6.5 again.

[0092] Example 27: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% gelatin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 2% sodium chloride, the second stabilizer is 0.1% casein, the preservative is 2% sodium azide, and the second surfactant is 0.2% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles was 50-100 nm, and the particle size of large-diameter latex particles was 300-420 nm. The concentration of MES buffer at pH 5.0 was 20 mM, the concentration of dual-diameter latex particles was 60 mg / L, the crosslinking agents were 12 mM EDC and 35 mM NHS, and the quencher was 0.2 M QSY-21. After the blocking reaction with human sST2 antibody was added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles was adjusted to 7.2. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 was 2 mg / mL, and the pH of reagent 2 was accurately adjusted to 7.2 again.

[0093] Example 28: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.6 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 2% sodium azide, and the second surfactant is 0.05% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction of human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 7.8. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 2 mg / mL, and the pH of reagent 2 is accurately adjusted to 7.8 again.

[0094] Example 29: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.6 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 2% sodium azide, the second surfactant is 0.1% alkyl glycoside, and the biochemical crosslinking agent is 0.01% BS3. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction of human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 2 mg / mL, and the pH of reagent 2 is accurately adjusted to 6.5 again.

[0095] Example 30: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.6 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 2% sodium azide, and the second surfactant is 0.2% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction of human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 7.2. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 0.5 mg / mL, and the pH of reagent 2 is accurately adjusted to 7.2 again.

[0096] Example 31: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% casein, the polyanionic agent is 0.9 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, and the accelerator is 0.01% PVP. The pH of reagent 1 is adjusted to 7.5. In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% casein, the preservative is 2% sodium azide, and the second surfactant is 0.05% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction of human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 7.8. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 0.5 mg / mL, and the pH of reagent 2 is accurately adjusted to 7.8 again.

[0097] Example 32: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% calcium chloride, the first stabilizer is 0.1% D-mannitol, the polyanionic agent is 0.9 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% ethyl cellulose, the preservative is 2% sodium azide, and the second surfactant is 0.1% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, and the particle size of large-diameter latex particles is 300-420 nm. The concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction with human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 0.5 mg / mL, and the pH of reagent 2 is accurately adjusted to 6.5 again.

[0098] Example 33: The components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% magnesium chloride, the first stabilizer is 0.1% ethyl cellulose, the polyanionic agent is 0.9 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% gelatin, the preservative is 2% sodium azide, and the second surfactant is 0.2% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction with human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 7.2. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 0.5 mg / mL, and the pH of reagent 2 is accurately adjusted to 7.2 again.

[0099] Example 34: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic polymer is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.1% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.1% alkyl glycoside.

[0100] Example 35: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic polymer is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.2% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.1% alkyl glycoside.

[0101] Regarding the preparation of reagent 2 in Examples 34-35, during the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles was 50-100 nm, the particle size of large-diameter latex particles was 300-420 nm, the concentration of MES buffer at pH 5.0 was 10 mM, the concentration of dual-diameter latex particles was 70 mg / L, the crosslinking agents were 12 mM EDC and 35 mM NHS, and the quencher was 0.05 M TCEP. After adding human sST2 antibody and blocking the reaction for 2 hours, the pH of the dilution solution of sST2 antibody-coated latex particles was adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 was 1.5 mg / mL, and the pH of reagent 2 was adjusted again to the final 6.5.

[0102] Example 36: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic polymer is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, and the accelerator is 0.1% PVP. The pH of reagent 1 is adjusted to 6.5. In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.1% alkyl glycoside.

[0103] Example 37: The components and concentrations of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic polymer is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.3% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.1% alkyl glycoside.

[0104] Regarding the preparation of reagent 2 in Examples 36-37, during the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles was 50-100 nm, the particle size of large-diameter latex particles was 300-420 nm, the concentration of MES buffer at pH 5.0 was 10 mM, the concentration of dual-diameter latex particles was 60 mg / L, and the crosslinking agents were 13 mM EDC and 40 mM NHS. After the crosslinking reaction, human sST2 antibody was directly added for blocking reaction for 2 hours. Then, the pH of the dilution solution of sST2 antibody-coated latex particles was adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 was 1.5 mg / mL, and the pH of reagent 2 was finally adjusted to 6.0.

[0105] Comparative Example 1: Compared with Example 1, the components, concentrations, and preparation process of the sST2 latex reagent are as follows: In Reagent 1, the first buffer is 15 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.4% PEG6000, and the pH of Reagent 1 is adjusted to 6.0; In Reagent 2, the second buffer is 15 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.05% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles was 100-150 nm, and the particle size of large-diameter latex particles was 300-420 nm. The concentration of MES buffer at pH 5.0 was 10 mM, the concentration of dual-diameter latex particles was 30 mg / L, the crosslinking agents were 15 mM EDC and 50 mM NHS, and the quencher was 0.05 M QSY-21. After the blocking reaction with human sST2 antibody was added for 3 hours, the pH of the dilution solution of sST2 antibody-coated latex particles was adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 was 0.75 mg / mL, and the pH of reagent 2 was finally adjusted to 6.5.

[0106] Comparative Example 2, compared with Example 10, the components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.01% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.3% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.3% sodium azide, and the second surfactant is 0.05% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 15 mM, the concentration of dual-diameter latex particles is 40 mg / L, the crosslinking agents are 14 mM EDC and 45 mM NHS, and the quencher is 0.15 M QSY-21. After adding human sST2 antibody and blocking reaction for 2 hours, the pH of the sST2 antibody-coated latex particle dilution solution is adjusted to 6.0. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 1.25 mg / mL, and the pH of reagent 2 is finally adjusted to 6.5.

[0107] Comparative Example 3, compared with Example 18, the components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 200 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% D-mannitol, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% lecithin, the promoter is 0.01% PEG6000, and the pH of reagent 1 is adjusted to 7; In reagent 2, the second buffer is 200 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.01% bovine serum albumin, the preservative is 3% sodium azide, and the second surfactant is 0.05% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 200-300 nm, the concentration of MES buffer at pH 5.0 is 15 mM, the concentration of dual-diameter latex particles is 50 mg / L, the crosslinking agents are 13 mM EDC and 40 mM NHS, and the quencher is 0.1 M QSY-21. After the blocking reaction with human sST2 antibody is added for 1.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 6.0. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 1.75 mg / mL, and the pH of reagent 2 is finally adjusted to 6.5.

[0108] Comparative Example 4, compared with Example 26, the components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% D-mannitol, the polyanionic agent is 0.1 mmol / L APAM, the preservative is 1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 7.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 4% sodium chloride, the second stabilizer is 0.1% casein, the preservative is 2% sodium azide, and the second surfactant is 0.01% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 20 mM, the concentration of dual-diameter latex particles is 60 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21. After the blocking reaction of human sST2 antibody is added for 2.5 hours, the pH of the dilution solution of sST2 antibody-coated latex particles is adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 2 mg / mL, and the pH of reagent 2 is accurately adjusted to 6.5 again.

[0109] Comparative Example 5, compared with Example 34, the components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.01% alkyl glycoside, the accelerator is 0.01% PVP, and the pH of reagent 1 is adjusted to 6.5; In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.1% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, the particle size of large-diameter latex particles is 300-420 nm, the concentration of MES buffer at pH 5.0 is 10 mM, the concentration of dual-diameter latex particles is 70 mg / L, the crosslinking agents are 12 mM EDC and 35 mM NHS, and the quencher is 0.05 M TCEP. After adding human sST2 antibody and blocking reaction for 2 hours, the pH of the sST2 antibody-coated latex particle dilution solution is adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 1.5 mg / mL, and the pH of reagent 2 is adjusted again to the final 6.5.

[0110] Comparative Example 6, compared with Example 36, the components, concentrations, and preparation process of the sST2 latex reagent are as follows: In reagent 1, the first buffer is 50 mmol / L HEPES buffer, the inorganic salt is 0.1% sodium chloride, the first stabilizer is 0.1% bovine serum albumin, the polyanionic agent is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, and there is no accelerator. The pH of reagent 1 is adjusted to 6.5. In reagent 2, the second buffer is 50 mmol / L MES buffer, the inorganic salt is 0.1% sodium chloride, the second stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% sodium azide, and the second surfactant is 0.1% alkyl glycoside. In the preparation of sST2 antibody-coated latex particles, the particle size of small-diameter latex particles is 50-100 nm, and the particle size of large-diameter latex particles is 300-420 nm. The concentration of MES buffer at pH 5.0 is 10 mM, the concentration of dual-diameter latex particles is 60 mg / L, and the cross-linking agents are 13 mM EDC and 40 mM NHS. After the cross-linking reaction, 0.05 M TCEP is added to quench the EDC for no more than 10 minutes. Then, human sST2 antibody is added according to the normal preparation process for blocking reaction for 2 hours. The pH of the diluent for sST2 antibody-coated latex particles is adjusted to 6.5. After adding the stock solution of reagent 2, the final concentration of sST2 antibody-coated latex particles in reagent 2 is 1.5 mg / mL, and the pH of reagent 2 is finally adjusted to 6.0.

[0111] Example 38: Performance evaluation of sST2 latex reagent Assay Method: The latex-enhanced immunoturbidimetric assay kits for detecting sST2 (hereinafter referred to as sST2 latex reagents) prepared in Examples 1-37 and Comparative Examples 1-4 were used for quantitative determination using a two-point endpoint method. The assay process was as follows: Three types of reaction tubes were set up, including blank tubes, sample tubes, and calibration tubes. 10 μL of purified water was added to the blank tube, 10 μL of the sample to be tested was added to the sample tube, and 10 μL of standard was added to the calibration tube. 150 μL of reagent 1 solution was added to each of the three reaction tubes and incubated at 37°C for 5 minutes. 50 μL of reagent 2 solution was then added to each reaction tube and mixed for 80 seconds. The absorbance (A1) of the three reaction tubes was read using a fully automated biochemical analyzer. After incubation at 37°C for 4 minutes, the absorbance (A2) of the three reaction tubes was read again using a fully automated biochemical analyzer. The measurement principle is based on the absorbance difference ΔA (i.e., a method to determine the concentration or activity of the analyte by measuring the total change in the concentration of the product or substrate from the start of the reaction to the point where the reaction reaches equilibrium). The calculation formula is as follows: ΔA = A2 - A1. The reaction tube setup and sample loading volume are shown in Table 1 below.

[0112] Table 1. Determination methods for sST2 latex reagents ; Sensitivity: The sensitivity of the sST2 latex reagent is mainly affected by the preparation process of the sST2 antibody-coated latex particles. A standard with a concentration of 54 ng / mL was used as the test sample. The sST2 latex reagents prepared in Examples 1-37, Comparative Examples 1-6, and third-party reagents were tested three times in the same sample tubes. The results of the absorbance difference between the sample tubes are shown in Table 2 below. The sensitivity was determined based on the absorbance difference (ΔAsample). The larger the difference, the higher the sensitivity.

[0113] Table 2 Sensitivity ; Table 2 shows the sensitivity test results. It can be seen that, while ensuring repeatability (a small coefficient of variation (CV%), i.e., CV% < 1%), the examples with relatively large mean differences are Examples 9, 11, 12, 18, 27, and 34. Firstly, all examples 1-35 involved a quenching process during the preparation of human sST2 antibody-coated latex particles. Secondly, in Examples 11, 12, 27, and 34, when selecting latex particles with dual particle sizes, the greater the difference between the small and large particle sizes, i.e., when latex particles with diameters of 50-100 nm and 300-420 nm are mixed, both relatively stable repeatability and high sensitivity with a relatively large difference can be obtained. In particular, Example 11 has the most stable repeatability (the smallest coefficient of variation, CV% = 0.13%). Further comparison of latex particle size and quencher type revealed that: Dual-size combination 1 (50-100nm and 300-420nm combinations in Examples 26-33) had a slightly more significant impact on improving sensitivity compared to dual-size combination 2 (100-150nm and 300-420nm combinations in Examples 1-9). However, compared to quencher group 1 (QSY-21 in Examples 26-33) and quencher group 1 (TCEP or no quencher in Examples 34-37), quencher group 1 showed a highly significant difference in its positive impact on improving sensitivity. Furthermore, QSY-21 was superior to TCEP, and TCEP was superior to no quencher. This indicates that the positive impact of the quencher is greater than the synergistic effect of the two latex particle sizes. In conclusion, the use of quenchers, the type of quencher, and the combination of dual-size latex particles can work synergistically to improve reagent sensitivity.

[0114] Linearity Range: Soluble growth-stimulating gene 2 recombinant protein was first added to MES buffer to prepare two samples with determined concentrations of high (432 ng / mL) and low (10 ng / mL). These two samples were then diluted in different proportions to prepare six different concentration samples with theoretical concentrations of 432 ng / mL, 216 ng / mL, 108 ng / mL, 54 ng / mL, 10 ng / mL, and 5 ng / mL. A total of 43 sST2 latex reagents and third-party reagents prepared according to the above 37 examples and 6 comparative examples were used to perform parallel measurements on these six different concentration samples. Each measurement was repeated three times, and the average value was taken as the detection result. Additionally, the injection volume at the highest point of 432 ng / mL was increased by 2 times and 3 times, and each measurement was repeated three times, with the average value taken as the detection result. The detection results are shown in Table 3 below.

[0115] Table 3 Measurement Results ; Table 3 shows that among the detection results in the range of 5 ng / mL to 432 ng / mL, only the correlation coefficients of the detection results of the three groups of sST2 latex reagents (Example 36, Comparative Example 1, and Comparative Example 2) are less than 0.975. That is, among the 43 sST2 latex reagents prepared in this invention, the correlation coefficients of 40 sST2 latex reagents in the range of 5 ng / mL to 432 ng / mL are relatively high, and the detection front band results of 2-fold and 3-fold high values ​​are also within the linear range. However, the correlation coefficients of the three sST2 latex reagents (Example 36 without quenching, r=0.9558), Comparative Example 1 with ultra-low concentration buffer, r=0.8784, and Comparative Example 2 with ultra-low concentration inorganic salt, r=0.9674) in the range of 5 ng / mL to 432 ng / mL are relatively low. A total of 31 sST2 latex reagents had a correlation coefficient greater than 0.99. Further comparison revealed that 8 of them still had a correlation coefficient greater than 0.999, namely: Example 3, Example 10, Example 11, Example 12, Example 22, Example 23, Example 29, and Example 31.

[0116] Three sets of comparative analysis: 1) Among the eight sST2 latex reagents with high correlation coefficients (r>0.999), Examples 3, 11, 12 and 29 were the ones with detection values ​​greater than 400 ng / mL for the customized sample with a high value (432 ng / mL). Further research on the preferred components and concentrations of these four examples revealed that: in Reagent 1, the polyanion was greater than 0.3 mmol / L APAM, the accelerator was 0.01% PVP, the surfactant was greater than 0.05% alkyl glycoside, and the stabilizer was 0.1% bovine serum albumin. It is speculated that polyanions, accelerators, surfactants and stabilizers have a significant impact on improving the linear range of reagent detection, especially on the detection accuracy of high values. 2) The extreme minimum detection value (78.5 ng / mL) of the customized sample with a high value (432 ng / mL) is Example 8. Although Example 8 also includes stabilizers with similar components and concentrations as described above (the relative coefficient of Example 8 is higher, r=0.9908), the polyanionic surfactant, the first surfactant, and the accelerator are different from the four preferred examples described above. It can be seen that the polyanionic surfactant, surfactant, and accelerator have a very important influence on improving the accuracy of high value detection. 3) Among the six comparative examples, except for Comparative Example 5, the detection values ​​of the other five comparative examples for the high-value (432 ng / mL) customized sample were relatively low (all ≤201.2 ng / mL). Therefore, a careful study of the components of Comparative Example 5, which significantly increased the detection value of the high value, revealed that Comparative Example 5 included the above-mentioned preferred components and concentrations of polyanion, promoter, and stabilizer (r=0.994). Compared with the above four preferred examples, the biggest difference of Comparative Example 5 is that the quencher in the preparation of sST2 antibody-coated latex particles is TCEP, and an ultra-low concentration of the first surfactant (0.01% alkyl glycoside) is used in Reagent 1. However, the detection value of the high value was relatively high (356.1 ng / mL), which further proves that including the same preferred components and concentrations of polyanion and promoter is of great significance for improving the accuracy of high-value detection. 4) In the preband detection results of 2 times and 3 times the high value concentration, the preband test values ​​of Examples 11, 12 and 29 can even reach 3 times the linear high value (432 ng / mL), which basically reaches the level of the magnetic particle chemiluminescence method represented by the third-party reagent. This can further expand the detection range of high values ​​and improve the detection accuracy of high values. In particular, BS3 was added as a biochemical cross-linking agent in reagent 2 of these three examples, which helps to further promote the detection value to reach 3 times the linear high value. This is most obvious in Example 11, and even significantly better than the third-party reagent.

[0117] The comparative analysis of the above three groups shows that the optimal composition and concentration of polyanionic surfactants, accelerators, and biochemical cross-linking agents have a significant impact on expanding the linear range of detection and improving the accuracy of high-value detection. Specifically, the preferred polyanionic surfactant is 0.3-0.6 mmol / L APAM, and the preferred accelerator is 0.01% PVP. Furthermore, the presence of 0.05%-0.1% alkyl glycosides in the surfactant also has a positive effect on improving the accuracy of high-value detection, while the preferred stabilizer, 0.1% bovine serum albumin, may have a positive impact on maintaining the linearity of the detection results. Table 3 also reveals that to ensure an error of less than 5% (95-105%) within the confidence interval for the high value (432 ng / mL), the detected value should be between 410 ng / mL and 453 ng / mL. Among the 37 examples and 6 comparative examples, only Examples 11, 12, and 29 meet this requirement. This shows that even with clinically common sST2 concentrations as high as 400 ng / mL, improving the detection accuracy of the high-value portion remains a major issue that needs optimization and resolution. At least to solve the technical problem of not being able to detect high sST2 concentrations, polyanionic polymers and promoters are key components.

[0118] In Table 3, the highest correlation coefficient (r) is for Example 11 (r = 0.9999). Based on the detection data of Example 11, with the theoretical concentration (xi) in the range of 5 ng / mL to 432 ng / mL as the independent variable and the mean value (yi) of the measurement results of Example 11 as the dependent variable, a linear evaluation result graph of Example 11 is plotted, as shown below. Figure 1 As shown, the regression equation corresponding to Example 11 is Y = 0.9635x - 1.6071 (R² + π / 2)². 2 =0.9998), indicating that there is a good linear relationship between the measured value and the theoretical value in Example 11, and the coefficient of determination (R²) is 0.9998. 2 The value is very close to 1, indicating that the detection value of Example 11 is reliable, has a very high degree of fit, and has a good linear relationship within the linear range of 5 ng / mL to 432 ng / mL.

[0119] Anti-interference ability: The sST2 latex reagents prepared in Examples 1-37 and Comparative Examples 1-6 were selected as the objects to be evaluated for anti-interference ability. A third-party reagent was used as a control. The test samples were from the same large number of human serum samples. Using the serum sample as a matrix, simulated test samples were prepared by adding rheumatoid factor RF, hemoglobin HB, and triglycerides TG. The above-mentioned sST2 latex reagents were used to conduct parallel comparisons of the test samples simulated by high-value interference. Each measurement was repeated 3 times, the mean was calculated and the relative deviation was calculated. The amount of rheumatoid factor RF, hemoglobin HB, and triglycerides TG added was 0 as a negative control for interference detection. The measurement results of the test samples simulated by the three types of interference (hereinafter referred to as interference samples) are shown in Table 4.

[0120] Table 4. Interference resistance of anti-interference devices

[0121] A detailed analysis of Table 4, comparing the detection results of high-value interfering substances (rheumatoid factor RF, hemoglobin HB, and triglycerides TG) in three types of serum samples, reveals that only the relative deviations of the detection values ​​of Examples 11, 15, 16, 29, Comparative Example 2, and the third-party reagent for these three types of interfering samples are less than 10%, and the average relative deviations are 2.75%, 5.12%, 6.02%, 6.86%, 5.77%, and 7.13%, respectively. This shows that the anti-interference performance of Example 11 is not only significantly better than other preferred examples and comparative examples, but also significantly better than the level of the magnetic particle chemiluminescence method represented by the third-party reagent. First, comparing the five sets of components and preparation processes found in Examples 11, 15, 16, 29, and Comparative Example 2, the following were observed: the preparation process of human sST2 antibody-coated latex particles; the pairing of latex particles (50-100 & 300-420); the quencher (QSY-21); the components and concentrations of buffer, stabilizer, and accelerator were identical; and the pH value of reagent 1 was 6.5. It is preliminarily inferred that the synergistic effect of human sST2 antibody-coated latex particles, buffer, stabilizer, accelerator, and pH value in improving the reagent's... The anti-interference ability has a significant impact; secondly, the anti-interference performance of Example 20 (with the largest average relative deviation of 17.64%) is significantly worse than that of other examples or comparative examples, and can be considered as the worst combination of anti-interference formulation components and concentrations. A comparison of Example 20 with the above three sets of examples with preferred anti-interference capabilities further reveals that the pairing of dual-size latex particles (50-100+300-420 & 50-100+200-300), buffer solution (HEPES+MES & phosphate), and stabilizer (BSA & D-) are crucial. The differences between mannitol, accelerator (polyvinylpyrrolidone & PEG6000), and pH values ​​(6.5 & 7) of reagent 1 were significant. Example 20, with its poor anti-interference ability, reversely verified that the pairing of dual-size latex particles in the preferred human sST2 antibody-coated latex particles, the synergistic effect of buffer, stabilizer, accelerator, and pH value of reagent 1 significantly improved the reagent's anti-interference ability. Third, among the five preferred anti-interference examples mentioned above, the relative deviation of all detection values ​​for these three types of interfering samples in Example 11 was less than [a certain value]. The mean relative deviation was 5% and the average deviation was significantly smaller (2.75%), which means that the anti-interference ability of the sST2 latex reagent prepared in Example 11 was significantly better than that of other examples or comparative examples. It can be used as the optimal combination of formulation components and concentrations. That is, the sST2 latex reagent prepared in Example 11 has a relatively strong anti-interference ability against chylomicrons or hemolyzed clinical samples containing high concentrations of rheumatoid factor (600 IU / mL), hemoglobin (800 IU / mL), and triglycerides (1.5%), and the accuracy of the measured values ​​is relatively reliable.

[0122] In summary, the analysis shows that the synergistic effect of the preferred components, concentrations, and preparation process of the sST2 latex reagent, including the preferred conditions of dual-size latex particle pairing, buffer solution, stabilizer, accelerator, and pH value, can significantly improve the reagent's interference resistance.

[0123] Stability: The stability of sST2 latex reagent was evaluated by storing Examples 1-37, Comparative Examples 1-6, and third-party reagents at 2-8°C for 0, 1, 3, 6, 12, 18, and 24 months, respectively, on the same test sample. The test samples were derived from the same serum sample, aliquoted, and stored at -80°C. Serum sST2 was measured randomly at the time of testing, with each measurement repeated three times. The linear change of the measured mean stability test value with storage time is shown below. Figure 2 As shown. First, the measured values ​​of the sST2 latex reagent prepared in Example 11, after being stored at 2-8℃ for 0, 1, 3, 6, 12, 18, and 24 months, showed relatively small changes with storage time, indicating the best stability. Its shelf life, at least, can be up to 24 months when stored at 2-8℃. The measured mean value of the third-party reagent also showed relatively small linear fluctuations with storage time, but the measured value after storage at 2-8℃ was slightly higher than the measured value before low-temperature storage. Second, Figure 2 Further comparison revealed that, compared to Example 11, the stability of the measured values ​​corresponding to Comparative Examples 1, 3, 4, and 5 showed highly significant differences with increasing storage time. Further analysis of the components of different sST2 latex reagents showed that: Comparative Example 1 used buffer solutions as low as 15 mmol / L in reagents 1 and 2; Comparative Example 3 used a very small amount of the second stabilizer in reagent 2; Comparative Example 4 added a very small amount of polyanion and used a very small amount of the second surfactant in reagent 2; and Comparative Example 5 used a very small amount of the first surfactant in reagent 1. This indicates that appropriate concentrations of buffer solutions, second stabilizers, polyanions, and surfactants all have a significant impact on maintaining the low-temperature stability of sST2 latex reagents. Third, Figure 2 It is evident that the measured value corresponding to Comparative Example 1 decreased significantly with increasing storage time, suggesting that an appropriate buffer concentration has a highly significant impact on maintaining the buffer environment for the specific binding reaction of antigen and antibody; relatively speaking, Figure 2Furthermore, it can be observed that the measured values ​​corresponding to Comparative Example 3 increased significantly with increasing storage time, suggesting that the appropriate concentration of the second stabilizer in Reagent 2 plays a significant role in preventing non-specific self-aggregation of antibody-coated latex particles. Fourth, the highly significant differences in the measured values ​​corresponding to Comparative Examples 4 and 5 further demonstrate that polyanions and surfactants also have a significant impact on maintaining the stability of latex reagents. Fifth, compared with Example 11, the measured values ​​of Examples 18-33 also showed significant differences with increasing storage time, further verifying that the preferred components and concentrations of buffer, stabilizer, polyanion, and surfactant all play an important role in maintaining the low-temperature stability of sST2 latex reagents. Finally, the measured values ​​corresponding to Example 11 in the stability evaluation results did not show significant fluctuations with increasing storage time, indicating optimal stability. This demonstrates that the components and proportions of Example 11 are relatively optimal. The stability evaluation further verifies the positive effects of the preferred components and concentrations of buffer, stabilizer, polyanion, and surfactant in Example 11 on maintaining the stability of the reagent. For example, when the first buffer is preferably 50 mmol / L HEPES buffer, the second buffer is preferably 50 mmol / L MES buffer, the first and second stabilizers are both preferably 0.1% bovine serum albumin, the polyanion is preferably 0.3 mmol / L APAM, and the first and second surfactants are both preferably 0.05% alkyl glycoside, the stability test results of the sST2 latex reagent are relatively optimal. In summary, the stability of the sST2 latex reagent is affected by multiple factors synergistically, mainly including buffer, stabilizer, polyanion, and surfactant.

[0124] Repeatability: The test samples were randomly selected large doses of clinical serum samples. The sST2 latex reagent prepared in 37 examples and 6 comparative examples of this invention and third-party reagents were used to measure the aliquoted test samples. Each measurement was repeated 10 times. The repeatability of the sST2 latex reagent was evaluated by calculating the mean and coefficient of variation (CV) of the measured values. The repeatability test results are shown in Table 5.

[0125] Table 5 Repeatability test results

[0126] Generally, a coefficient of variation (CV) of less than 10% is considered acceptable for repeatability, indicating good repeatability of the reagent test. Table 5 shows that the average values ​​of the 37 examples and 6 comparative examples were not significantly different (all less than 10%). Twelve examples had a CV of less than or equal to 2.5%, with Example 11 (0.3 mmol / L APAM) showing the lowest CV (0.7%) among repeated test results, making it the only representative with a CV less than 1%. This demonstrates that the sST2 latex reagent prepared in Example 11 exhibits highly significant and beneficial repeatability.

[0127] Correlation: Based on the performance verification results above, the sensitivity, linear range, low-temperature storage stability, and repeatability of the sST2 latex reagent prepared in Example 11 of this invention are relatively superior. Therefore, the sST2 latex reagent prepared in Example 11 and a third-party reagent were used to conduct parallel tests on the same test samples to evaluate the correlation of Example 11. The test samples were 60 randomly selected clinical serum samples. Correlation curves were plotted using the measured values ​​of the two reagents on the same test sample, with the x-axis and y-axis respectively. Figure 3 As shown, the regression equation is y = 1.0021x - 0.5823 (R²). 2 = 0.9938), which shows that the measurement results of the two reagents are highly consistent and exhibit good linear correlation. That is, the measured values ​​of the sST2 latex reagent prepared in Example 11 have good consistency and good accuracy and correlation.

[0128] Performance indicators of the sST2 latex reagents prepared in 37 examples and 6 comparative examples show that: the present invention optimized the preferred components and concentrations of buffer, stabilizer, polyanion, surfactant, and accelerator in reagents 1 and 2. Performance evaluation results demonstrate that: whether a quencher is used during the preparation of sST2 antibody-coated latex particles, the type of quencher, and the combination of dual-size latex particles have a synergistic and positive impact on improving the sensitivity of the sST2 latex reagent; the preferred components and concentrations of polyanion and biochemical cross-linking agent have a positive impact on expanding the linear range of detection and improving the accuracy of high-value detection; due to the diversity of interfering substances, the present invention at least demonstrates that the preferred conditions of dual-size latex particle pairing, buffer, stabilizer, accelerator, and pH value have a synergistic and positive impact on improving the interference resistance of the reagent; the preferred conditions of buffer, stabilizer, polyanion, biochemical cross-linking agent, and surfactant have a synergistic and positive impact on improving the stability of the reagent. Furthermore, the repeatability detection data corresponding to the 37 examples and 6 comparative examples of the present invention all meet the requirements. Furthermore, preliminary experiments of this invention revealed that when using latex-enhanced immunoturbidimetry to determine soluble growth-stimulating gene 2 protein, higher concentrations of inorganic salts were detrimental to improving detection sensitivity and repeatability. Therefore, the inorganic salt concentration was significantly reduced to the range of 0.1%-0.2%. With the synergistic effect of optimization of other key components, the overall performance of the reagent was greatly improved. In particular, the sST2 latex reagent prepared in Example 11 exhibited relatively superior sensitivity, anti-interference ability, stability, repeatability, and correlation with third-party reagents. Moreover, existing common detection methods for soluble growth-stimulating gene 2 (sST2) protein cannot meet the requirements of greater than 4... The detection requirements for high concentrations of sST2 above 00 ng / mL, especially the phenomenon that the quantitative detection value of high sST2 cannot be reached when using latex-enhanced immunoturbidimetric assay, highlight the crucial role of the preferred components and concentrations of the polyanion, promoter, and biochemical cross-linking agent creatively added in Example 11 of this invention in expanding the linear detection range and improving the accuracy of high-value detection. The evaluation results of its comprehensive performance indicators not only meet or exceed those of third-party reagents, but also demonstrate that the performance of the preferred latex reagent of this invention can reach the level of magnetic microparticle chemiluminescence method, making it particularly suitable for monitoring or evaluating the severity of heart failure at high sST2 concentrations.

[0129] Specific embodiments of the present invention have been described in detail, making them readily understandable to those skilled in the art. However, based on all the disclosed descriptions, different or similar modifications or substitutions may be made to certain specific details, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A reagent composition for determining soluble growth-stimulating gene 2 protein using latex-enhanced immunoturbidimetry, comprising reagent 1 and reagent 2, which are independent of each other; Reagent 1 has a pH of 6.0–7.5 and contains a first buffer solution, inorganic salts, a first stabilizer, a polyanionic agent, a preservative, a first surfactant, and an accelerator; Reagent 2 has a pH of 6.0–7.8 and contains a second buffer solution, inorganic salts, a second stabilizer, a preservative, a second surfactant, a biochemical cross-linking agent, and soluble growth-stimulating gene 2 protein antibody-coated latex particles; the final concentration of inorganic salts in both Reagent 1 and Reagent 2 is 0.1%–2%; in, In reagent 1, the polyanion is selected from any one of polyanionic cellulose, polymaleic acid, APAM and chondroitin sulfate at a concentration of 0.3 to 0.9 mmol / L; the accelerator is selected from any one or two of PEG6000 and PVP at a concentration of 0.01% to 0.3%. In reagent 2, the concentration of the soluble growth-stimulating gene 2 protein antibody-coated latex particles in reagent 2 is 0.5–2.0 mg / mL. The latex particles used to prepare the soluble growth-stimulating gene 2 protein antibody-coated latex particles are selected from dual-size latex particles, which include small-size latex particles and large-size latex particles. The particle size range of the small-size latex particles is 50–150 nm, and the particle size range of the large-size latex particles is 200–420 nm. The preparation of the soluble growth-stimulating gene 2 protein antibody-coated latex particles includes the use of organic compounds as quenching agents.

2. The reagent composition according to claim 1, characterized in that, Both the first buffer and the second buffer are selected from any one or two of Tris-HCl buffer, phosphate buffer, HEPES buffer and MES buffer in the range of 50 to 280 mmol / L, and the main components of the first buffer and the second buffer are different. The inorganic salt is selected from any one or two of calcium chloride, sodium chloride, and magnesium chloride; The first stabilizer and the second stabilizer are selected from any one or two of bovine serum albumin, mannitol, gelatin compounds, casein and CE series at a concentration of 0.1% to 0.5%; The preservative is selected from any one or two of sodium azide, benzoic acid, ProClin-300, and antibiotics; and the final concentration of the preservative in reagent 1 or reagent 2 is 0.1% to 3%. The first surfactant is selected from any one or two of the following: alkyl glycosides, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipin, sophorolipid, and A90, at a concentration of 0.05% to 0.2%. The second surfactant is selected from any one or two of the following: alkyl glycosides, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipin, sophorolipid, A90, and P-40, at a concentration of 0.05% to 0.2%. In the dual-size latex particles, the particle size range of the small-size latex particles is selected from 50-150 nm, and the particle size range of the large-size latex particles is selected from 200-420 nm.

3. The reagent composition according to claim 2, characterized in that, The first buffer is HEPES buffer; the first stabilizer is bovine serum albumin; the polyanionic surfactant is APAM; the first surfactant is an alkyl glycoside; and the promoter is PVP.

4. The reagent composition according to claim 2, characterized in that, The second buffer is MES buffer; the second stabilizer is bovine serum albumin; the second surfactant is alkyl glycoside; the concentration of the soluble growth-stimulating gene 2 protein antibody-coated latex particles in reagent 2 is 0.75–1.75 mg / mL, and the particle size range of the large-diameter latex particles is selected from 300–420 nm.

5. A soluble growth-stimulating gene 2 protein assay kit, characterized in that, The reagent composition comprises any one of claims 1-4, and further comprises a quality control sample and a calibrator, wherein the quality control sample and the calibrator each contain at least two concentration levels of recombinant protein resistant to soluble growth-stimulating gene 2, and the buffers are MES buffers containing human serum.

6. The soluble growth-stimulating gene 2 protein assay kit according to claim 5, characterized in that, The quality control material contains at least two concentration levels of the protein responsible for resisting soluble growth-stimulating gene 2 expression, and these two concentration levels are as follows: Level 1: 20 ng / mL~60 ng / mL; Level 2: 80 ng / mL~130 ng / mL; The calibrator contains at least six concentration levels of soluble growth-stimulating gene 2 protein: 0 ng / mL, 10 ng / mL, 54 ng / mL, 108 ng / mL, 216 ng / mL, and 432 ng / mL.

7. A method for preparing latex particles coated with a soluble growth-stimulating gene 2 protein antibody, characterized in that, Includes the following steps: S1: Take the small-diameter latex particles and large-diameter latex particles from the reagent composition according to any one of claims 1-4, suspend the dual-diameter latex particles in 10-20 mM MES buffer, and make the concentration of the dual-diameter latex particles 30-70 mg / L, and the pH value of the MES buffer is 5. S2: Add EDC and NHS, and perform a crosslinking reaction at room temperature for 1 hour; S3: Add 0.05-0.2M quencher to quench EDC for no more than 10 minutes to terminate the catalytic activity of unreacted EDC; S4: Add an equal molar amount of human soluble growth-stimulating gene 2 protein antibody to the two-particle-size latex particles, and block the reaction at room temperature for 1.5-3 hours to obtain a crude solution containing latex particles coated with soluble growth-stimulating gene 2 protein antibody. S5: The crude solution containing soluble growth-stimulating gene 2 protein antibody-coated latex particles is subjected to low-temperature high-speed centrifugation or filtration to remove the supernatant, thereby obtaining the stock solution of soluble growth-stimulating gene 2 protein antibody-coated latex particles. S6: Dilute the soluble growth-stimulating gene 2 protein antibody-coated latex particles to 10 g / L again using 10-20 mM, pH 5.0 MEM buffer, adjust the pH to 6.0-7.8, and obtain the diluted solution of soluble growth-stimulating gene 2 protein antibody-coated latex particles; aliquot and store at 4℃ for later use.

8. The method for preparing latex particles coated with soluble growth-stimulating gene 2 protein antibody according to claim 7, characterized in that, In step S1: the particle size of the small-diameter latex particles is 50-100nm, the particle size of the large-diameter latex particles is 300-420nm, and the concentration of the dual-diameter latex particles is 30-70mg / L. In step S2: the concentration of EDC is 12-15 mM, and the concentration of NHS is 35-50 mM; In step S3: the quenching agent can be either QSY-21 or TCEP.

9. A method for preparing a soluble growth-stimulating gene 2 protein assay kit, characterized in that, The reagent composition according to any one of claims 1-4 is prepared, wherein, The preparation process of reagent 1 is as follows: take the first buffer solution and add it to the container, and stir at a speed of 300-400 rpm; then add the inorganic salt, the first stabilizer, the polyanionic agent, the preservative, the first surfactant and the accelerator in the components of reagent 1 to the first buffer solution in order of the predetermined components and their concentrations and mix well; finally, use a pH adjuster to adjust the pH of reagent 1 to a pH value of 6.0-7.5, and thus prepare reagent 1; The preparation process of reagent 2 is as follows: First, add the second buffer solution to a container, then add the inorganic salt, the second stabilizer, the preservative, and the second surfactant to the second buffer solution in sequence according to the predetermined components and their concentrations, and mix well. Adjust the pH value to 6 to complete the preparation of the reagent 2 stock solution. Add the above reagent 2 stock solution to the diluent of the soluble growth-stimulating gene 2 protein antibody-coated latex particles according to any one of claims 7-8, and further dilute the soluble growth-stimulating gene 2 protein antibody-coated latex particles to a final concentration of 0.5-2.0 mg / mL, and mix well. Finally, adjust the pH to the range of 6.0-7.8 to obtain reagent 2.

10. The reagent composition according to any one of claims 1-4, the soluble growth-stimulating gene 2 protein assay kit according to any one of claims 5-6, the method for preparing soluble growth-stimulating gene 2 protein antibody-coated latex particles according to claims 7-8, and the application of the method for preparing the soluble growth-stimulating gene 2 protein assay kit according to claim 9 in the preparation of latex reagent-related products.

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