A soluble growth stimulating expression gene 2 protein assay kit, preparation method and application

Abstract

The application provides a soluble growth stimulating expression gene 2 protein determination kit, a preparation method and an application, and belongs to the technical field of biochemical immunoassay. The application adopts a latex enhanced immunoturbidimetry to detect the soluble growth stimulating expression gene 2 protein, optimizes and adjusts key components of reagent 1 and reagent 2, obtains optimal components and concentrations of polyanions, surfactants, accelerators and biochemical cross-linking agents, solves technical problems such as poor stability of sST2 related reagents and incapability of meeting high value detection accuracy of sST2 greater than 400 ng / mL, and the optimal components and concentrations can significantly improve the detection accuracy of high values in a linear range of 5 ng / mL to 432 ng / mL, in particular, the prepared sST2 latex reagent has sensitivity, anti-interference ability, stability, repeatability and pre-band reaching and being superior to third-party reagents, and can well meet the demand of clinical use.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical immunoassay, and particularly relates to a soluble stimulating expression gene 2 protein determination kit, and a preparation method of the soluble stimulating expression gene 2 protein determination kit and application thereof in biochemical immunoassay. BACKGROUND

[0002] The stimulating expression gene 2 (ST2) is located on human chromosome 2, and the ST2 protein (about 40 kb) encoded by the stimulating expression gene 2 is a member of the interleukin-1 receptor family, directly affects the main subtypes of transmembrane type (ST2L) and soluble type (sST2) in the progress of heart disease, and the two subtypes are formed by selective splicing and can be specifically combined with the functional ligand IL-33. The ST2L contains three extracellular immunoglobulin-like domains, a transmembrane domain and a short cytoplasmic tail, the sST2 is a soluble form of the ST2L, lacks the transmembrane domain and the cytoplasmic tail, and contains a unique 9-amino acid C-terminal sequence. The structure is prone to conformational changes due to environmental factors, so the stability of the structure is poor in vitro storage. When the body is stimulated by inflammation or tissue damage, the related cells will induce the expression of sST2. As one of the important biomarkers of the heart, the natriuretic peptide only reflects the hemodynamic state, while the sST2 reflects fibrosis and remodeling, and the sST2 concentration is not interfered by factors such as age, gender, race, BMI index, kidney function, has a higher specificity, and is closely related to the progress of heart disease. For example, under normal circumstances, the sST2 concentration is low (if the immunofluorescence dry quantitative method is used for detection, the concentration value is less than 35.0 ng / mL), and if heart failure occurs, the sST2 concentration significantly increases, and the detection value can be higher than 400 ng / mL.

[0003] At present, the common detection methods of the marker soluble growth stimulating expression gene 2 (sST2) protein in the clinic or laboratory are colloidal gold immunochromatography, fluorescence immunochromatography, chemiluminescence immunoassay and enzyme-linked immunoassay. Although the latex immunoturbidimetry has high sensitivity, short detection time and simple operation, it is less common. The latex enhanced immunoturbidimetry is a kind of latex immunoturbidimetry with wider application and higher sensitivity. Its basic principle is that when the specific antibody is fixed and coated on the latex particles, and the specific antigen in the sample reacts with the specific antibody, larger complex particles are formed by aggregation, and the turbidity value of these larger particles is significantly amplified at a specific wavelength. Because the turbidity value is linearly related to the amount of antigen and antibody, the standard curve drawn by comparison can more sensitively calculate the quantitative concentration of the target substance (such as sST2 protein) in the sample. The sST2 protein is detected by the latex enhanced immunoturbidimetry, and there are still many interference factors, such as the non-specific binding of common substances such as rheumatoid factor, triglyceride and hemoglobin in the sample to be detected to the latex reagent, which may cause false positive or false negative results. In addition, the common technical problems in the immunodetection of sST2 protein include narrow linear range, low high-value detection accuracy, poor stability of reagent kit, etc. For example, the magnetic microparticle enzyme chemiluminescence method (publication number CN110208549A) has a detection range of 1-300 ng / ml, which still cannot meet the detection needs of sST2 with a concentration higher than 400 ng / ml. Although the immunochromatographic test strip invention patent (CN118311272A) discloses a linear range of 1.00-460.80 ng / mL, the average value of three high-value detection results (407.74) is only 88.49% accurate to the reference concentration (460.80 ng / mL), and the high-value detection accuracy is still low. Therefore, the present application provides a latex enhanced immunoturbidimetry reagent kit for detecting sST2, a preparation method thereof and an application thereof, which has a wider detection range, higher sensitivity, higher stability, higher accuracy and higher reliability. SUMMARY

[0004] In order to overcome the above problems, the present application provides a latex enhanced immunoturbidimetry reagent kit for detecting soluble growth stimulating expression gene 2 protein, which has significantly improved performance indicators such as detection linear range, sensitivity, anti-interference ability, stability, repeatability and accuracy, as well as a preparation method and application thereof.

[0005] In order to achieve the above-mentioned purposes, the present application realizes the following technical solutions:

[0006] In a first aspect, the present application provides a reagent composition for determining soluble growth stimulating expressed gene 2 protein by using latex-enhanced immunoturbidimetry, comprising reagent 1 and reagent 2 independently; the pH of the reagent 1 is 6.0-7.5, and the reagent 1 comprises a first buffer, inorganic salt, first stabilizer, polyanion, preservative, first surfactant and promoter; the pH of the reagent 2 is 6.0-7.8, and the reagent 2 comprises a second buffer, inorganic salt, second stabilizer, preservative, second surfactant, biochemical cross-linking agent and latex particles coated with soluble growth stimulating expressed gene 2 protein antibody; the final concentration of inorganic salt in the reagent 1 and the reagent 2 is 0.1%-2%; wherein, in the reagent 1, the polyanion is selected from any one of 0.3-0.9 mmol / L polyanion cellulose, polymaleic acid, APAM and chondroitin sulfate; the promoter is selected from any one or both of 0.01%-0.3% PEG6000 and PVP; in the reagent 2, the concentration of the latex particles coated with soluble growth stimulating expressed gene 2 protein antibody in the reagent 2 is 0.5-2.0 mg / mL, and the latex particles in the preparation of the latex particles coated with soluble growth stimulating expressed gene 2 protein antibody are selected from double-size latex particles, wherein the double-size latex particles comprise small-size latex particles and large-size latex particles; the particle size of the small-size latex particles ranges from 50 nm to 150 nm, and the particle size of the large-size latex particles ranges from 200 nm to 420 nm; and the process for preparing the latex particles coated with soluble growth stimulating expressed gene 2 protein antibody comprises using an organic compound as a quencher.

[0007] According to a preferred embodiment of the present application, in the polyanion, the Chinese name of APAM is polyacrylamide, and the English full name is Anionic Polyacrylamide.

[0008] According to a preferred embodiment of the present application, in the promoter, the Chinese name of PEG6000 is polyethylene glycol 6000, and the English full name is Polyethylene Glycol 6000.

[0009] According to a preferred embodiment of the present application, in the promoter, the biochemical cross-linking agent is preferably BS3, the Chinese name of which is bis(sulfosuccinimidyl) suberate sodium salt, and the English full name is Bis(sulfosuccinimidyl) suberate sodium salt.

[0010] According to a preferred embodiment of the present application, in the promoter, the Chinese name of PVP is polyvinyl pyrrolidone, and the English full name is Polyvinyl Pyrrolidone.

[0011] According to a preferred embodiment of the present application, 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 or two of 0.1%-0.5% bovine serum albumin, mannitol, gelatin compound, 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 final concentration of the preservative is 0.1%-3%; the first surfactant is selected from any one or two of 0.05%-0.2% alkyl glycoside, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipid, sophorolipid, A90; the second surfactant is selected from any one or two of 0.05%-0.2% alkyl glycoside, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipid, sophorolipid, A90, P-40; the particle size range of the small particle size latex particles is selected from 50-150 nm, and the particle size range of the large particle size latex particles is selected from 200-420 nm.

[0012] Preferably, in the first buffer and the second buffer, the Chinese name of the HEPES buffer is 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and the English full name of HEPES is 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid.

[0013] Further preferably, the buffer capacity of the HEPES buffer for adjusting PH is 6.0-7.5, and the concentration is 15-150 mm.

[0014] Preferably, in the first buffer and the second buffer, the Chinese name of the MES buffer is 2-(N-morpholine) ethanesulfonic acid buffer, and the English full name of MES is 2-(N-Morpholino)ethanesulfonic acid.

[0015] Further preferably, the buffer capacity of the MES buffer for adjusting PH is 6.0-7.8, and the concentration is 15-150 mm.

[0016] Preferably, in the first stabilizer and the second stabilizer, the Chinese name of BSA is bovine serum albumin, and the English full name of BSA is Bovine Serum Albumin.

[0017] Preferably, among the first stabilizer and the second stabilizer, the Chinese name of the CE series is cellulose ether (Cellulose Ether) series, and the preferred representative is ethyl cellulose (Ethyl Cellulose, abbreviated as EC).

[0018] Preferably, the surfactant includes the first surfactant in reagent 1 and the second surfactant in reagent 2; wherein the preferred representative of the Triton series is Triton X-100, also known as Triton X-100, or polyoxyethylene octyl phenyl 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-octyl phenoxy polyethoxy ethanol; the chemical name of SDS is sodium dodecyl sulfate, and the full English name is Sodium Dodecyl Sulfate; the chemical name of A90 is polyoxyethylene biphenylene styrenated phenyl ether, and the full English name is Polyoxyethylene biphenylene styrenated phenyl ether, and the product name is EMULGEN A-90; the chemical name of B66 is polyoxyethylene tribenzyl phenyl ether, and the full English name is Polyoxyethylenetribenzyl phenyl ether, and the product name is EMULGEN B-66; P-40 belongs to a non-ionic surfactant, and the chemical name is nonyl phenyl polyethylene glycol, and the full English name is Nonylphenyl-polyethyleneglycol.

[0019] Further preferably, the surfactant is an alkyl glycoside.

[0020] Preferably, the inorganic salt in reagent 1 is sodium chloride and / or calcium chloride.

[0021] Further preferably, the 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 is also conducive to reducing the viscosity of polyanions.

[0022] Preferably, the preservative in reagent 1 and reagent 2 is sodium azide.

[0023] 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 polyanion is APAM or polymaleic acid.

[0024] Further preferably, the first buffer is a 15-150 mm HEPES buffer and a MES buffer.

[0025] Further preferably, the second buffer is a 15-150 mm MES buffer and a phosphate buffer.

[0026] Further preferably, the pH value of the reagent 1 is 6.5.

[0027] Further preferably, the pH value of the reagent 2 is 6.5.

[0028] Further preferably, the inorganic salt is sodium chloride.

[0029] Further preferably, the first stabilizer and the second stabilizer are bovine serum albumin.

[0030] Further preferably, the preservative is 0.1-3.0% sodium azide.

[0031] Further preferably, the first surfactant and the second surfactant are alkyl glycosides.

[0032] Further preferably, in the reagent 1, the first buffer is a HEPES buffer; the first stabilizer is bovine serum albumin; the polyanion is APAM; the first surfactant is alkyl glycoside; and the accelerator is PVP.

[0033] Further preferably, in the reagent 2, the second buffer is a MES buffer; the second stabilizer is bovine serum albumin; the second surfactant is alkyl glycoside; and the concentration of the soluble growth stimulating expression gene 2 protein antibody coated latex particles in the reagent 2 is 0.75-1.75 mg / mL, and the particle size range of the large particle size latex particles is selected from 300-420 nm.

[0034] In a second aspect, the present application provides a soluble growth stimulating expression gene 2 protein assay kit, comprising the above-mentioned reagent composition, further comprising a quality control and a calibration, both of which contain at least two concentration levels of anti-soluble growth stimulating expression gene 2 recombinant protein, and the buffer is a MES buffer containing human serum.

[0035] According to one preferred embodiment of the present application, in the soluble growth stimulating expression gene 2 protein assay kit, the quality control product contains at least two concentration levels of soluble growth stimulating expression gene 2 recombinant protein, and the two concentration levels are: 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 expression gene 2 recombinant protein, and the six concentration levels are: 0 ng / mL, 10 ng / mL, 54 ng / mL, 108 ng / mL, 216 ng / mL and 432 ng / mL.

[0036] In a third aspect, the present application provides a preparation method of soluble growth stimulating expression gene 2 protein antibody-coated latex particles, comprising the following steps: S1: taking double-particle-size latex particles suspended in 10-20 mM MES buffer, so that the concentration of the double-particle-size latex particles is 30-70 mg / L; S2: adding EDC and NHS, and reacting at room temperature for 1 hour to catalyze the formation of an amide bond between the carboxyl group on the surface of the latex particles and the amino group on the antibody; S3: using 0.05-0.2 M quenching agent to quench the EDC for no more than 10 minutes to terminate the catalytic activity of the unreacted EDC; S4: adding human soluble growth stimulating expression gene 2 protein antibodies in an equimolar amount to the double-particle-size latex particles, and closing the reaction at room temperature for 1.5-3 hours to obtain a crude solution containing soluble growth stimulating expression gene 2 protein antibody-coated latex particles; S5: removing the supernatant from the crude solution containing soluble growth stimulating expression gene 2 protein antibody-coated latex particles by low-temperature high-speed centrifugation or filtration to obtain a stock solution of soluble growth stimulating expression gene 2 protein antibody-coated latex particles; S6: diluting the soluble growth stimulating expression gene 2 protein antibody-coated latex particles to 10 g / L using 10-20 mM MEM buffer, adjusting the pH to 6.0-7.8, and obtaining a dilution solution of soluble growth stimulating expression gene 2 protein antibody-coated latex particles; and storing the dilution solution at 4°C for use.

[0037] According to one preferred embodiment of the present application, in step S1 of the preparation method of the soluble growth stimulating expression gene 2 protein antibody-coated latex particles, the pH value of the 10-20 mM MES buffer is 5.

[0038] According to one preferred embodiment of the present application, in the preparation method of the soluble growth stimulating expression gene 2 protein antibody coated latex particles, in the S1 step: the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, and the final concentration of the equimolar mixture of the small particle size latex particles and the large particle size latex particles is 30-70 mg / L; in the S2 step: the concentration of EDC is 12-15 mM, and the concentration of NHS is 35-50 mM; in the S3 step: the quencher is an organic compound, which is any one of QSY-21 or TCEP.

[0039] In a fourth aspect, the present application provides a preparation method of a soluble growth stimulating expression gene 2 protein assay kit, which is used to prepare the above-mentioned reagent composition, wherein the reagent 1 configuration process is: taking the first buffer into a container and stirring at a rotation speed of 300-400 rpm; then adding the inorganic salt, the first stabilizer, the polyanion, the preservative, the first surfactant and the accelerator in the reagent 1 component into the first buffer according to the predetermined components and their concentrations, and mixing uniformly, and finally adjusting the pH of the reagent 1 to 6.0-7.5 by using the pH adjuster, so as to obtain the reagent 1; the reagent 2 configuration process is: first taking the second buffer into a container, and then adding the inorganic salt, the second stabilizer, the preservative and the second surfactant into the second buffer according to the predetermined components and their concentrations, mixing uniformly, and adjusting the pH value to 6.0-7.8, so as to complete the preparation of the reagent 2 stock solution; adding the reagent 2 stock solution into the diluent of the soluble growth stimulating expression gene 2 protein antibody coated latex particles prepared above, further diluting the soluble growth stimulating expression gene 2 protein antibody coated latex particles to a final concentration of 0.5-2.0 mg / mL, and mixing uniformly; and finally adjusting the pH to 6.0-7.8, so as to obtain the reagent 2.

[0040] According to one preferred embodiment of the present application, in the preparation method of the soluble growth stimulating expression gene 2 protein assay kit, the final concentration of the soluble growth stimulating expression gene 2 protein antibody coated latex particles is 0.75-1.75 mg / mL.

[0041] In a fifth aspect, the present application also claims the application of the above-mentioned reagent composition, the soluble growth stimulating expression gene 2 protein assay kit, the preparation method of the soluble growth stimulating expression gene 2 protein antibody coated latex particles and the preparation method of the soluble growth stimulating expression gene 2 protein assay kit in the preparation of latex reagent related products.

[0042] The beneficial effects of the present application are:

[0043] The application provides a detection method for detecting soluble growth stimulating expression gene 2 protein by using latex enhanced immune turbidimetry, an immune detection kit, a preparation method and application thereof.

[0044] The application mainly adopts the latex enhanced immune turbidimetry for detection, and optimizes components, concentrations and preparation processes of reagent 1 and reagent 2, for example, the polyanion in the reagent 1 is preferably polyacrylamide (APAM), and a promoter is additionally added, a surfactant is preferably alkyl glycoside, and the promoter is preferably polyvinylpyrrolidone (PVP), through mutual synergistic cooperation between the selected double-particle-size latex particles, the preferred buffer, the stabilizer and the promoter, and the preferred conditions of the reagent pH value, the ability of resisting interference of chylomicron particles is significantly improved.

[0045] In the optimization process of the sST2 latex reagent provided by the application, regarding the buffer, the first buffer is preferably HEPES buffer, because the HEPES buffer can effectively resist external pH changes through the protonation / deprotonation reaction of the two nitrogen atoms on the piperazine ring and the acid-base equilibrium ability of the sulfonic acid group (R-SO3H), so that the reagent is more stable. Regarding the stabilizer, it is generally believed that ionic stabilizers (such as SDS, etc.) can effectively stabilize the latex at a concentration of 0.1-1%, but may cause flocculation when the concentration exceeds 2%; and non-ionic stabilizers (such as bovine serum albumin BSA, etc.) can maintain the stability of the latex at a concentration of 0.5-2%, but may cause particle aggregation at a concentration of 5%; however, the pre-experiment of the application found that whether it is an ionic stabilizer or a non-ionic stabilizer, a concentration exceeding 0.5% is not conducive to maintaining the stability of the latex, and finally a low concentration of non-ionic stabilizer in the range of 0.1-0.5%, such as bovine serum albumin, is preferably selected as the stabilizer. Regarding the polyanion, the application preferably selects APAM as the component, which is presumably because APAM can promote specific immune reactions between sST2 antibody-coated latex particles and sST2 antigens in the sample, accelerate the aggregation of particles, and inhibit the increase of signal-to-noise ratio and the occurrence of non-specific reactions. Regarding the accelerator, the application preferably selects PVP in a low concentration range of 0.01-0.3%, which can shield the charge neutralization effect of electrolytes or proteins on the latex in the serum sample, prevent false aggregation, and in addition, the carbonyl group on the PVP molecule can form a hydrogen bond with the carboxyl group of the antibody, assist the antibody to be specifically bound on the surface of the sST2 antibody-coated latex particles, improve the specific antibody-antigen binding efficiency (improve the specific aggregation rate between the latex particles and the sST2 antigen), and ensure that the turbidity change is linearly related to the concentration of the sST2 antigen in the sample to be measured, which directly improves the sensitivity of the detection, expands the linear detection range, and improves the detection accuracy of high values, and also provides a positive effect in the anti-interference ability. Regarding the biochemical crosslinking agent, the addition of the biochemical crosslinking agent, bis-succinimidyl suberate sodium salt (BS3), in the reagent 2 can form an amide bond through the NHS ester group and the primary amine of the protein, so that the performance of the reagent is more stable, and more importantly, the application can still maintain linearity in a high multiple front band (such as 2-3 times high value), that is, the detection line of high value concentration is obviously improved by 2 times and 3 times, which shows that the sST2 latex reagent with the preferred component effectively avoids the front band effect, can measure higher concentration samples, and further widens the detection range and improves the upper limit of detection.The conventional latex reagent is affected by the front band effect, and high concentration samples exceeding the linear range need to be pre-diluted, which may introduce errors and increase operation time. However, by introducing BS3, the limitation of the front band effect can be broken through, and high multiple front band samples can be directly detected, which not only reduces the sample dilution step, reduces human error, but also improves the detection efficiency, especially suitable for accurate monitoring of diseases such as inflammation or heart failure with abnormally high sST2 concentration, and more timely detection of abnormal sST2 concentration information.

[0046] As for inorganic salts, the purpose is to adjust the ionic strength of the reagent and ensure that the reaction system can be in a suitable osmotic pressure environment to avoid self-aggregation of latex particles or antibody separation due to osmotic pressure imbalance; preferably sodium chloride or calcium chloride, and at least containing sodium chloride, because sodium chloride can effectively avoid the interference of specific binding reaction between sST2 in the sample to be tested and sST2 antibody coated latex particles, and at the same time, sodium chloride is beneficial to reduce the viscosity of polyanion. It is also found in the screening process of the present application that the inclusion of calcium chloride in the inorganic salt is beneficial to solving the hook effect, which is presumably due to the fact that Ca 2+ Through charge neutralization, it preferentially promotes antigen-antibody specific binding, which to some extent helps to solve the problem of high concentration sST2 detection value. In reagent 1 and reagent 2, the optimal concentration of inorganic salt depends on the nature of the detection object, the surface properties of the latex particles, the requirements of the reaction system, and the properties of the inorganic salt itself. If the detection object is a small molecule antigen or antibody, or the latex particles need to be aggregated as soon as possible, and the inorganic salt itself is compatible with other components of the reaction system, the concentration of the inorganic salt can be appropriately increased. For example, in the patent (CN119395307A), when the detection object is SAA, it is found that high concentration of inorganic salt (such as NaCl) can shield the charge on the surface of the latex particles, thereby promoting particle aggregation to improve detection sensitivity. However, the effect of inorganic salt concentration on detection performance needs to be verified by experiment. Due to the complexity and diversity of components in the latex reagent, blindly applying the empirical value of the commonly used inorganic salt concentration is ineffective. In the present application, due to the instability of the anti-soluble growth stimulating expression gene 2 (sST2) protein antibody and the inherent technical problem of high value detection accuracy, the present application selects human sST2 antibody to coat double-particle-size latex particles. In order to ensure the stability between double-particle-size latex particles, the inorganic salt concentration screening experiment found that a lower 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 concentration in reagent 2 is higher (4%), the fluctuation of the detection value 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 the sST2 latex reagent.

[0047] The preparation process of the sST2 latex reagent is optimized, especially the preparation process of the sST2 antibody-coated latex particles, and the double-particle-size latex particles and the addition of a quencher are preferred, so that the sensitivity and linear range of the prepared sST2 latex reagent are significantly improved, and the key performance indicators such as anti-interference, stability and repeatability of the kit are also improved. Among them, the double-particle-size latex particles are two particle sizes of carboxyl microspheres, the small particle size latex microspheres (50-150 nm) have more carboxyl groups and a large specific surface area, can bind a large amount of antibodies, and are beneficial to improve the linear range of the reagent; the large particle size latex microspheres (200-420 nm) can capture low-concentration antibodies and enhance low-value precision. In the latex particle coating process, an organic compound is used as a quencher to further improve the performance of the sST2 latex reagent. The quencher terminates the catalytic activity of the unreacted activator (EDC), prevents the unbound activator from continuing to react with the latex particle surface or the subsequently added antibody / antigen, improves the specificity and stability of the sST2 antibody-coated latex particles for detection, and at the same time, the quenching reaction helps to ensure that the antibody can be specifically bound to the surface of the latex particles without being interfered by the unreacted activator, achieving the purpose of optimizing the coating effect. Especially, the quencher belongs to an organic compound, which has the advantages of high stability, good compatibility and low toxicity. The application preferably uses QSY-21 as a quencher, and experimental results show that the addition of a quencher is beneficial to improve the sensitivity, linear range and stability of the sST2 latex reagent.

[0048] There are many components in the latex reagent, and the preferred combination of components has a positive synergistic effect on improving the overall performance of the reagent. For example: in terms of sensitivity, whether to use a quencher in the preparation process of the sST2 antibody-coated latex particles, the type of quencher, and the combination of double-particle-size latex particles have a synergistic effect and a positive influence on improving the sensitivity of the sST2 latex reagent. In terms of linear range, the pairing of double-particle-size latex particles, and the preferred components and concentrations of added polyanions, accelerators and biochemical cross-linking agents have a positive influence on expanding the linear range of detection and improving the accuracy of high-value (3 times of 432 ng / mL) detection. In terms of anti-interference, the pairing of double-particle-size latex particles, buffer, stabilizer, accelerator and pH value also have a synergistic effect and a positive influence on improving the interference ability of the reagent. In addition, the preferred conditions of buffer, stabilizer, polyanion and surfactant also have a synergistic effect and a positive influence on improving the stability of the reagent.

[0049] The performance indexes of the sST2 latex reagent provided by the application have achieved significant progress as a whole. By optimizing the five main components of the buffer, inorganic salt, stabilizer, surfactant and preservative in the reagent 1 and the reagent 2, and creatively adding polyanion, accelerator and biochemical crosslinking agent in the reagent 1, and by pairing and combining the double-particle-size latex particles, introducing the quenching process and adding biochemical crosslinking agent in the reagent 2 to optimize the preparation process of the soluble growth stimulating expression gene 2 protein antibody coated latex particles, the stability of the chemical coupling of the double-particle-size latex particles and the soluble growth stimulating expression gene 2 protein antibody is improved, and the sensitivity, linear range and anti-interference ability of the detection of the sST2 protein are further improved. In addition, the sST2 latex reagent prepared by the application also shows better performance in stability, repeatability and the like. It is verified by specific experiments that the detection results of the preferred embodiment 11 of the application and the third party reagent are highly correlated. The application not only provides a specific sST2 latex reagent, but also constructs a more optimized latex enhanced immunoturbidimetry detection platform for sST2 protein and a preparation method of a latex enhanced immunoturbidimetry reagent for soluble growth stimulating expression gene 2 protein. Through comprehensive performance evaluation and specific clinical sample experiment verification, the detection sensitivity, specificity, linear range, anti-interference ability, stability, repeatability and accuracy of the sST2 latex reagent for high-value sST2 protein detection are significantly improved. The coefficient of variation CV% of the sensitivity detection value can be 0.13%, the linear detection range can be 5 ng / mL-432 ng / mL, the anti-rheumatoid factor (RF) interference concentration can be as high as 600 mmol / L, the anti-hemoglobin (HB) interference concentration can be as high as 800 mmol / L, the anti-triglyceride (TG) interference concentration can be as high as 1.5%, the sST2 latex reagent can be stably placed at 2-8°C for 24 months, the detection value is basically stable after long-term low-temperature storage, the coefficient of variation between 10 repeated determinations can be 0.7%, and these performance indexes all reach and are better than the level of the third party reagent (high-sensitivity magnetic microparticle chemiluminescence method). The coincidence rate between the measured values of the preferred embodiment 11 and the third party reagent is still good (R 2 = 0.9938), that is, the detection results of the sST2 latex reagent provided by the application are highly linearly correlated with the gold standard (chemiluminescence), and are suitable for preliminary screening of clinical auxiliary detection. It can be seen that the sST2 latex reagent provided by the application not only has the advantages of simple operation, low cost, good stability, rapid detection and good repeatability of the latex reagent, but also reaches the level of high sensitivity and wide linear range specific to the magnetic microparticle chemiluminescence method.

[0050] The application provides a soluble growth stimulating expression gene 2 protein determination kit, a preparation method and application thereof, and optimizes key components such as buffer, inorganic salt, stabilizer, surfactant and preservative in reagent 1 and reagent 2 and the preparation process of the soluble growth stimulating expression gene 2 protein antibody coated latex particles, through optimization of the preparation process of the soluble growth stimulating expression gene 2 protein determination kit, and through specific experiments and verification of clinical samples, it is proved that the sST2 latex reagent provided by the application can significantly improve the comprehensive performance index of the latex enhanced immunoturbidimetry for determining the soluble growth stimulating expression gene 2 protein, especially solve the technical problems of narrow linear range of sST2 detection and difficulty in measuring high-concentration sST2, and overall improve the sensitivity, anti-interference ability, stability, repeatability, accuracy and other performance indexes of the sST2 latex reagent, and the sST2 latex reagent can realize automatic detection in cooperation with a full-automatic biochemical analyzer, and is particularly suitable for monitoring or evaluating the severity of heart failure under high sST2 concentration. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 A linear evaluation result graph of sST2 latex reagent prepared for the embodiment 11 of the application and detection results of third-party reagents, the abscissa is the theoretical value, the ordinate is the measured value, and the unit is ng / mL; S11 represents the embodiment 11, and A represents the third-party reagent;

[0052] Figure 2 A stability evaluation result graph of sST2 latex reagents prepared for 37 groups of embodiments and 6 groups of comparative examples of the application, wherein A represents the third-party reagent, S1-S37 respectively represent 37 groups of embodiments, and D1-D6 respectively represent 6 groups of comparative examples; the abscissa is the storage time under the condition of 2-8 DEG C, and the unit is month; the ordinate is the quantitative determined concentration value, and the unit is ng / mL;

[0053] Figure 3 A correlation evaluation result graph between the measured values of the sST2 latex reagent prepared for the embodiment 11 of the application and the third-party reagent, the abscissa represents the measured value of the embodiment 11, the unit is ng / mL, and the ordinate represents the measured value of the third-party reagent, the unit is ng / mL. DETAILED DESCRIPTION

[0054] The exemplary embodiments of the application are described in detail below with reference to the accompanying drawings, wherein the same or similar notations represent the same or similar concepts, such as soluble growth stimulating expression gene 2 protein = sST2 protein = sST2. In the following detailed description, many specific details are set forth in order to provide a thorough understanding of the embodiments of the present disclosure. The following embodiments are only used to illustrate the application, and should not be regarded as limiting the scope of the application.

[0055] The experimental methods used in the following examples are conventional methods, and the materials, reagents, etc. used are commercially available unless otherwise specified. Among them, the soluble growth stimulating expression gene 2 recombinant protein (product number: LA446) is purchased from Medix Biochemica Company; the anti-human soluble growth stimulating expression gene 2 protein antibody (human soluble growth stimulating expression gene 2 protein antibody, referred to as human sST2 antibody, product number: 100686) is purchased from Medix Biochemica Company; A90 and B66 are purchased from the non-ionic surfactant EMULGEN series products (EMULGEN A-90 and EMULGEN B-66) of Huawang Company; QSY-21 (carboxylic acid succinimidyl ester, which belongs to amine reactive quencher, product number: Q20132) is purchased from ThermoFisher Company; alkyl glycoside (product number: A864768) is purchased from Macklin Company; latex particles are purchased from Jetway Biotech (Beijing) Biotechnology Co., Ltd.; the activators EDC (product number: N808856) and NHS (product number: N811124) are purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; the full-automatic biochemical analyzer is purchased from Hitachi Company, Japan, and the model number is 7180; the third-party reagent is an sST2 determination kit (magnetic microparticle chemiluminescence method) purchased from Beijing Li De Man Biochemical Co., Ltd. However, it is obvious that one or more embodiments can be implemented without these specific details, and the specific conditions are not specified in the examples. The molecular biology experimental methods not specified in the following examples are performed according to the specific methods listed in the book “Molecular Cloning Laboratory Guide” (third edition) by J. Sambrook, or according to the kit and product instructions.

[0056] The application object of the determination in the present application refers to the body fluid biological sample collected from human or animal body. After the sample collection work is completed, the biological sample has been separated from the living human or animal body. For example, blood samples (whole blood / serum / plasma), body fluids, tissues, excreta, isolated cultures (blood cultures, sputum cultures, etc.) all belong to in vitro non-living biological samples. The determination process is completed in vitro. The direct purpose of the determination is to determine whether the target protein (soluble growth stimulating expression gene 2 protein) or its amino acids from the degradation products of the target protein exists in the sample. The determination result is helpful for the doctor to judge the information of the inquiry. The determination belongs to the determination of components or contents in in vitro non-living biological samples. There is no process of directly obtaining the diagnosis result of the disease or the health condition in the determination. Therefore, the present application does not belong to the disease diagnosis method, which meets the basic requirements of the patent protection object of the “Patent Law”. The specific embodiments of the present application are described below.

[0057] The present application optimizes the components, concentrations and configuration process of reagent 1 and reagent 2 which are independent of each other in the latex-enhanced immunoturbidimetric assay for soluble growth stimulating expression gene 2 (sST2) protein. Regarding the key components, reagent 1 includes a first buffer, inorganic salt, first stabilizer, polyanion, preservative, first surfactant and accelerator; reagent 2 includes a second buffer, inorganic salt, second stabilizer, preservative, second surfactant and soluble growth stimulating expression gene 2 protein antibody coated latex particles (sST2 antibody coated latex particles for short).

[0058] Among them, the first buffer and the second buffer are preferably any one or two of 50-150 mmol / L Tris-HCl buffer, phosphate buffer, MES buffer and HEPES buffer, and preferably the main components of the first buffer and the second buffer are different; the inorganic salt is preferably any one or two of calcium chloride, sodium chloride and magnesium chloride, and the preferred concentration of the inorganic salt in reagent 1 and reagent 2 is 0.1%-2%; the first stabilizer and the second stabilizer are preferably any one or two of 0.1%-0.5% bovine serum albumin, mannitol, gelatin compound, casein and CE series; the preservative is preferably any one or two of 0.1%-3% sodium azide, benzoic acid, ProClin-300 and antibiotic series; the first surfactant and the second surfactant are preferably any one or two of 0.05%-0.2% alkyl glycoside, lecithin, Tween 20, Triton series (preferably Triton X-100), sodium dodecyl sulfate (SDS), polyoxyethylene tribenzyl phenyl ether (B66), rhamnolipid, sophorolipid, A90 (polyoxyethylene lauryl ether, preferably EMULGEN A90 of Showa Denko K.K.). Reagent 1 also includes: 0.3-0.9 mmol / L of polyanion, and the polyanion is selected from any one of polyanionic cellulose, polymaleic acid, polyacrylamide (APAM) and chondroitin sulfate; 0.01%-0.3% accelerator, and the accelerator is selected from any one of polyethylene glycol 6000 (PEG6000) and polyvinylpyrrolidone (PVP); the second surfactant in reagent 2 can also be selected from any one or two of 0.05%-0.2% alkyl glycoside, lecithin, Tween 20, Triton series, SDS, B66, rhamnolipid, sophorolipid, A90 and nonyl phenyl polyethylene glycol (P-40).

[0059] The preparation method of the soluble growth stimulating expression gene 2 protein assay kit of the present application comprises the following steps: first, preparing reagent 1, then, based on the preparation of reagent 2 stock solution, preparing the double-size latex particles coated with anti-human soluble growth stimulating expression gene 2 protein antibody (referred to as soluble growth stimulating expression gene 2 protein antibody coated latex particles, or sST2 antibody coated latex particles), then, preparing reagent 2, and finally, according to the specifications, preparing reagent 1, reagent 2, quality control or standard in one kit.

[0060] The preparation method of reagent 1 is as follows: first, adding the first buffer into a container and stirring at a speed of 300-400 rpm, then, adding inorganic salt, first stabilizer, polyanion, preservative, first surfactant and accelerator into the buffer according to the predetermined components and concentrations, and finally, adjusting the pH of reagent 1 to 6-7.5 by using a pH adjuster, thereby obtaining reagent 1.

[0061] The preparation method of reagent 2 stock solution is as follows: first, adding the second buffer into a container and stirring at a speed of 300-400 rpm, then, adding inorganic salt, second stabilizer, preservative, biochemical crosslinking agent and second surfactant into the second buffer according to the predetermined components and concentrations, and finally, adjusting the pH to 6.0-7.8 by using a pH adjuster, thereby obtaining reagent 2 stock solution.

[0062] The preparation process of the sST2 antibody-coated latex particles is as follows: 1) according to a volume ratio of 1:1, small particle size (50-100 nm or 100-150 nm) latex particles and large particle size (200-300 nm or 300-420 nm) latex particles are taken respectively, and after being mixed with 10-20 mM, pH 5.0 MES buffer, the concentration of the double particle size latex particles is 30-70 mg / L; 2) 12-15 mM EDC and 35-50 mM NHS are added, and the cross-linking reaction is carried out at room temperature (20-25 °C) 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) 0.1-0.25 M quencher (QSY-21 or TCEP) is added, and the EDC is quenched for no more than 10 minutes; 4) human sST2 antibody is added in an equal molar amount to the double particle size latex particles, and the closed reaction is carried out at room temperature (20-25 °C) for 1.5-3 hours on a shaking table to obtain a crude solution containing sST2 antibody-coated latex particles; 5) the supernatant is removed from the above-mentioned crude solution by low-temperature high-speed centrifugation or filtration, and the original solution of sST2 antibody-coated latex particles is obtained; 6) the sST2 antibody-coated latex particles are diluted to 10 g / L using 10-20 mM, pH 5.0 MEM buffer, and the pH is adjusted to 6.0-7.8 to obtain a dilution solution of sST2 antibody-coated latex particles. In the preparation process, the purpose of quenching is to react the active ester group, amino group and other organic functional groups on the quencher with the active groups of EDC, so that EDC loses catalytic ability. In the quencher, the chemical name of QSY-21 is carboxylic acid succinimidyl ester, and its English full name is QSY21 carboxylic acid, NHS Ester; the chemical name of TCEP is tris (2-carboxyethyl) phosphine, and its English is Tris (2-carboxyethyl) phosphine.

[0063] The configuration method of reagent 2 is as follows: in the dilution solution of sST2 antibody-coated latex particles, the above-mentioned prepared reagent 2 stock solution is added, and the sST2 antibody-coated latex particles are further diluted to a final concentration of 0.5-2.0 mg / mL (preferably, the concentration range is 0.75-1.75 mg / mL), and after being mixed, the pH is adjusted to 6.5-7.8, and reagent 2 is prepared.

[0064] In the latex-enhanced immunoturbidimetric method for detecting sST2 kit (referred to as sST2 latex reagent), the volume ratio of reagent 1 and reagent 2 is preferably 4:1, and the quality control product or standard product is prepared from human serum containing soluble growth stimulating expression gene 2 recombinant protein or similar serum matrix liquid, wherein the content of human serum is not less than 5%. The specific configuration process of the quality control product is as follows: soluble growth stimulating expression gene 2 recombinant protein antigen is added to MES buffer to prepare two levels of quality control products, wherein the target value range of level 1 is 20-60 ng / mL, and the target value range of level 2 is 80-130 ng / mL. The specific configuration process of the standard product is as follows: soluble growth stimulating expression gene 2 recombinant protein is mixed in 15-100 mmol / L MES buffer to prepare five concentration series, and the target concentration of soluble growth stimulating expression gene 2 recombinant protein is 432 ng / mL, 216 ng / mL, 108 ng / mL, 54 ng / mL and 10 ng / mL respectively; in addition, the standard product series also includes a human serum standard product without soluble growth stimulating expression gene 2 recombinant protein, i.e. the concentration of soluble growth stimulating expression gene 2 recombinant protein is 0 ng / mL. 5-10% human serum is added to the standard product with six series concentrations of 0 ng / mL, 10 ng / mL, 54 ng / mL, 108 ng / mL, 216 ng / mL and 432 ng / mL.

[0065] The present application sets 37 groups of examples including the above-mentioned core components, concentrations and preparation methods in the overall experimental design of the latex-enhanced immunoturbidimetric method for detecting sST2 kit, aiming to screen the preferred components and concentrations of different buffers, inorganic salts, stabilizers, preservatives and surfactants, and also includes verifying the addition of polyanion and promoter in reagent 1, and the combination of different size particle diameter double particle size latex particles in reagent 2, and the optimization of the preparation process of sST2 antibody coated latex particles by adding quenching agent, so as to improve the specificity and sensitivity of the reagent. In addition, six comparative examples are further set, aiming to verify the limit use concentration of buffer, inorganic salt, stabilizer, polyanion, surfactant and promoter, especially by comparing the optimal concentration ratio of polyanion and surfactant to improve the best synergistic effect of the performance index of the latex-enhanced immunoturbidimetric method for detecting sST2 kit.

[0066] The key components, concentrations and preparation processes between the 37 groups of examples and the 6 groups of comparative examples of the latex-enhanced immunoturbidimetric method for detecting sST2 kit are as follows; and the reagent kits prepared by using these examples and comparative examples are used to verify their respective linear range, sensitivity, anti-interference ability, stability and repeatability and other related performance indexes, and the specific detection results are as follows.

[0067] The components and concentrations of the sST2 latex reagent of Example 1 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 polyanion 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 value 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.

[0068] The components and concentrations of the sST2 latex reagent of Example 2 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 polyanion 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 value 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.

[0069] The components and concentrations of the sST2 latex reagent of Example 3 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 polyanion 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 value 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.

[0070] The components and concentrations of the sST2 latex reagent of Example 4 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 polyanion 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.

[0071] The components and concentrations of the sST2 latex reagent of Example 5 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 polyanion 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.

[0072] The components and concentrations of the sST2 latex reagent of Example 6 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 polyanion 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.

[0073] 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 polyanion is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% A90, the promoter is 0.01% PVP, and the pH value 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.

[0074] 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 polyanion is 0.3 mmol / L polymaleic acid, the preservative is 3% sodium azide, the first surfactant is 0.05% A90, the promoter is 0.01% PEG6000, and the pH value 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.

[0075] 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 polyanion is 0.3 mmol / L APAM, the preservative is 3% sodium azide, the first surfactant is 0.05% A90, the promoter is 0.01% PVP, and the pH value 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.

[0076] For the preparation of reagent 2 of Examples 1-9, in the preparation of sST2 antibody-coated latex particles, the particle size of small-sized latex particles was 100-150 nm, the particle size of large-sized latex particles was 300-420 nm, the concentration of MES buffer at pH 5.0 was 10 mM, the concentration of double-sized 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 blocking reaction for 3 hours by adding human sST2 antibody, the pH of the diluent of sST2 antibody-coated latex particles was adjusted to 6.5, reagent 2 stock solution was added to make the final concentration of sST2 antibody-coated latex particles in reagent 2 be 0.75 mg / mL, and the pH of reagent 2 was finally adjusted to 6.5.

[0077] Example 10, the components and concentrations of sST2 latex reagent were as follows: in reagent 1, the first buffer was 50 mmol / L HEPES buffer, the inorganic salt was 0.1% sodium chloride, the first stabilizer was 0.1% bovine serum albumin, the polyanion was 0.3 mmol / L APAM, the preservative was 0.3% sodium azide, the first surfactant was 0.05% alkyl glycoside, the accelerator was 0.01% PVP, and the pH of reagent 1 was adjusted to 6.5; in reagent 2, the second buffer was 50 mmol / L MES buffer, the inorganic salt was 1% sodium chloride, the second stabilizer was 0.1% bovine serum albumin, the preservative was 0.3% sodium azide, and the second surfactant was 0.05% alkyl glycoside.

[0078] Example 11, the components and concentrations of sST2 latex reagent were as follows: in reagent 1, the first buffer was 50 mmol / L HEPES buffer, the inorganic salt was 0.1% sodium chloride, the first stabilizer was 0.1% bovine serum albumin, the polyanion was 0.3 mmol / L APAM, the preservative was 0.3% sodium azide, the first surfactant was 0.05% alkyl glycoside, the accelerator was 0.01% PVP, and the pH of reagent 1 was adjusted to 6.5; in reagent 2, the second buffer was 50 mmol / L MES buffer, the inorganic salt was 2% sodium chloride, the second stabilizer was 0.1% bovine serum albumin, the preservative was 0.3% sodium azide, the second surfactant was 0.05% alkyl glycoside, and the biochemical crosslinking agent was 0.01% BS3.

[0079] 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 polyanion 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 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.

[0080] 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 polyanion 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 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.

[0081] 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 polyanion 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 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.

[0082] 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 polyanion 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 salt is 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.

[0083] 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 polyanion 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 salt is 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.

[0084] 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 polyanion is 0.3 mmol / L polymaleic acid, the preservative is 0.3% sodium azide, the first surfactant is 0.05% sophorolipid, the accelerator is 0.01% PEG6000, 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 0.4% 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.

[0085] For the preparation of Reagent 2 of Examples 10-17, in the preparation of sST2 antibody-coated latex particles, the particle size of small-sized latex particles was 50-100 nm, the particle size of large-sized latex particles was 300-420 nm, the concentration of MES buffer at pH 5.0 was 15 mM, the concentration of double-sized latex particles was 40 mg / L, the crosslinking agent was 14 mM EDC and 45 mM NHS, and the quenching agent was 0.15 M QSY-21; after blocking reaction for 2 hours by adding human sST2 antibody, the pH of the diluent of sST2 antibody-coated latex particles was adjusted to 6.0, and after adding the stock solution of Reagent 2, the sST2 antibody-coated latex particles were allowed to react at a final concentration of 1.25 mg / mL of Reagent 2, and the pH of Reagent 2 was finally adjusted to 6.5.

[0086] Example 18, the components and concentrations of sST2 latex reagent were as follows: in Reagent 1, the first buffer was 200 mmol / L HEPES buffer, the inorganic salt was 0.1% sodium chloride, the first stabilizer was 0.1% D-mannitol, the polyanion was 0.3 mmol / L APAM, the preservative was 0.1% sodium azide, the first surfactant was 0.05% lecithin, the accelerator was 0.01% PEG6000, and the pH of Reagent 1 was adjusted to 7; in Reagent 2, the second buffer was 200 mmol / L MES buffer, the inorganic salt was 0.1% sodium chloride, the second stabilizer was 0.3% bovine serum albumin, the preservative was 3% sodium azide, and the second surfactant was 0.05% alkyl glycoside.

[0087] Example 19, the components and concentrations of sST2 latex reagent were as follows: in Reagent 1, the first buffer was 100 mmol / L HEPES buffer, the inorganic salt was 0.1% sodium chloride, the first stabilizer was 0.1% gelatin, the polyanion was 0.3 mmol / L APAM, the preservative was 0.1% sodium azide, the first surfactant was 0.05% Tween 20, the accelerator was 0.01% PEG6000, and the pH of Reagent 1 was adjusted to 7; in Reagent 2, the second buffer was 200 mmol / L MES buffer, the inorganic salt was 0.1% sodium chloride, the second stabilizer was 0.5% bovine serum albumin, the preservative was 3% sodium azide, and the second surfactant was 0.05% alkyl glycoside.

[0088] 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 salt is 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 polyanion 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.

[0089] 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 salt is 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 polyanion 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.

[0090] 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 salt is 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 polyanion 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.

[0091] 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 salt is 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 polyanion 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.

[0092] Example 24, the components and concentrations of the sST2 latex reagent are as follows: in reagent 1, the first buffer is 200 mmol / L phosphate buffer, the inorganic salt is 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 polyanion 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 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.

[0093] 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 salt is 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 polyanion 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.

[0094] For the preparation of Reagent 2 of Examples 18-25, in the preparation of sST2 antibody-coated latex particles, the particle size of small-sized latex particles was 50-100 nm, the particle size of large-sized latex particles was 200-300 nm, the concentration of pH 5.0 MES buffer was 15 mM, the concentration of double-sized latex particles was 50 mg / L, the crosslinking agent was 13 mM EDC and 40 mM NHS, and the quenching agent was 0.1 M QSY-21; after blocking reaction with human sST2 antibody for 1.5 hours, the pH of the diluent of sST2 antibody-coated latex particles was adjusted to 6.0, and 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.

[0095] For the preparation of Reagent 2 of Examples 18-25, in the preparation of sST2 antibody-coated latex particles, the particle size of small-sized latex particles was 50-100 nm, the particle size of large-sized latex particles was 200-300 nm, the concentration of pH 5.0 MES buffer was 15 mM, the concentration of double-sized latex particles was 50 mg / L, the crosslinking agent was 13 mM EDC and 40 mM NHS, and the quenching agent was 0.1 M QSY-21; after blocking reaction with human sST2 antibody for 1.5 hours, the pH of the diluent of sST2 antibody-coated latex particles was adjusted to 6.0, and 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.

[0096] The components, concentrations and preparation process of the sST2 latex reagent of Example 27 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 polyanion 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, 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double-particle-size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21; after adding the human sST2 antibody blocking reaction for 2.5 hours, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 7.2, reagent 2 stock solution is added, the final concentration of the sST2 antibody-coated latex particles in reagent 2 is 2 mg / mL, and the pH of reagent 2 is accurately adjusted to 7.2 again.

[0097] The components, concentrations and preparation process of the sST2 latex reagent of Example 28 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 polyanion 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, 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double-particle-size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21; after blocking reaction for 2.5 hours by adding human sST2 antibody, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 7.8, reagent 2 stock solution is added, the final concentration of the 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.

[0098] The components, concentrations and preparation process of the sST2 latex reagent of Example 29 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 polyanion 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, 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double-particle-size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21; after adding the human sST2 antibody blocking reaction for 2.5 hours, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 6.5, reagent 2 stock solution is added, the final concentration of the 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.

[0099] The components, concentrations and preparation process of the sST2 latex reagent of Example 30 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 polyanion 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, 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double-particle-size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21; after adding the human sST2 antibody blocking reaction for 2.5 hours, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 7.2, reagent 2 stock solution is added, the final concentration of the 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.

[0100] The components, concentrations and preparation process of the sST2 latex reagent of Example 31 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 polyanion 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, 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double-particle-size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quenching agent is 0.2 M QSY-21; after blocking reaction for 2.5 hours by adding human sST2 antibody, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 7.8, reagent 2 stock solution is added, the final concentration of the 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.

[0101] The components, concentrations and preparation process of the sST2 latex reagent of Example 32 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 polyanion 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, 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double-particle-size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quencher is 0.2 M QSY-21; after adding the human sST2 antibody blocking reaction for 2.5 hours, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 6.5, reagent 2 stock solution is added, the final concentration of the 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.

[0102] The components and concentrations of the sST2 latex reagent of Example 33 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 polyanion 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double-particle-size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quenching agent is 0.2 M QSY-21; after adding the human sST2 antibody blocking reaction for 2.5 hours, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 7.2, reagent 2 stock solution is added, the final concentration of the 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.

[0103] The components and concentrations of the sST2 latex reagent of Example 34 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 polyanion 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.

[0104] 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 polyanion 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.

[0105] For the preparation of reagent 2 of Examples 34-35, in the preparation of sST2 antibody-coated latex particles, the particle size of small particle size latex particles is 50-100 nm, the particle size of large particle size latex particles is 300-420 nm, the concentration of pH 5.0 MES buffer is 10 mM, the concentration of double particle size latex particles is 70 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quenching agent is 0.05 M TCEP; after adding human sST2 antibody blocking reaction for 2 hours, the pH of the diluent of sST2 antibody-coated latex particles is adjusted to 6.5, and after adding reagent 2 stock solution, 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 to the final 6.5 again.

[0106] 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 polyanion is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, the accelerator is 0.1% 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.

[0107] The components and concentrations of the sST2 latex reagent of Example 37 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 polyanion 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, 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.

[0108] For the preparation of reagent 2 of Examples 36-37, in the preparation of sST2 antibody-coated latex particles, the particle size of small particle size latex particles is 50-100 nm, the particle size of large particle size latex particles is 300-420 nm, the concentration of pH 5.0 MES buffer is 10 mM, the concentration of double particle size latex particles is 60 mg / L, the crosslinking agent is 13 mM EDC and 40 mM NHS, after the crosslinking reaction, human sST2 antibody is directly added for blocking reaction for 2 hours, then the pH of the sST2 antibody-coated latex particle diluent is adjusted to 6.5, reagent 2 stock solution is added to make the final concentration of sST2 antibody-coated latex particles in reagent 2 be 1.5 mg / mL, and the pH of reagent 2 is finally adjusted to 6.0.

[0109] Comparative Example 1, in comparison with Example 1, the components, concentrations and preparation process of 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 polyanion 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 process of sST2 antibody-coated latex particles, the particle size of small particle size latex particles is 100-150 nm, the particle size of large particle size latex particles is 300-420 nm, the concentration of pH 5.0 MES buffer is 10 mM, the concentration of double particle size latex particles is 30 mg / L, the crosslinking agent is 15 mM EDC and 50 mM NHS, and the quencher is 0.05 M QSY-21; after adding human sST2 antibody blocking reaction for 3 hours, the pH of the diluent of sST2 antibody-coated latex particles is adjusted to 6.5, reagent 2 stock solution is added to make the final concentration of sST2 antibody-coated latex particles in reagent 2 be 0.75 mg / mL, and the pH of reagent 2 is finally adjusted to 6.5.

[0110] Comparative Example 2, in contrast to Example 10, the components, concentrations and preparation process of 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 polyanion 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 process of sST2 antibody-coated latex particles, the particle size of small particle size latex particles is 50-100 nm, the particle size of large particle size latex particles is 300-420 nm, the concentration of pH 5.0 MES buffer is 15 mM, the concentration of double particle size latex particles is 40 mg / L, the crosslinking agent is 14 mM EDC and 45 mM NHS, and the quencher is 0.15 M QSY-21; after adding human sST2 antibody blocking reaction for 2 hours, the pH of the diluent of sST2 antibody-coated latex particles is adjusted to 6.0, reagent 2 stock solution is added to make the final concentration of sST2 antibody-coated latex particles in reagent 2 be 1.25 mg / mL, and the pH of reagent 2 is finally adjusted to 6.5.

[0111] Comparative Example 3, in contrast to Example 18, the components, concentrations and preparation process of 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 polyanion is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% lecithin, 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 0.01% bovine serum albumin, the preservative is 3% sodium azide, and the second surfactant is 0.05% alkyl glycoside. In the preparation process of sST2 antibody-coated latex particles, the particle size of small particle size latex particles is 50-100 nm, the particle size of large particle size latex particles is 200-300 nm, the concentration of pH 5.0 MES buffer is 15 mM, the concentration of double particle size latex particles is 50 mg / L, the crosslinking agent is 13 mM EDC and 40 mM NHS, and the quencher is 0.1 M QSY-21; after adding human sST2 antibody blocking reaction for 1.5 hours, the pH of the diluent of sST2 antibody-coated latex particles is adjusted to 6.0, reagent 2 stock solution is added, and 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.

[0112] 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 polyanion 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 process of the sST2 antibody-coated latex particles, the particle size of the small particle size latex particles is 50-100 nm, the particle size of the large particle size latex particles is 300-420 nm, the concentration of the pH 5.0 MES buffer is 20 mM, the concentration of the double particle size latex particles is 60 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quenching agent is 0.2 M QSY-21; after the blocking reaction for 2.5 hours after adding the human sST2 antibody, the pH of the sST2 antibody-coated latex particle diluent is adjusted to 6.5, reagent 2 stock solution is added, the final concentration of the 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.

[0113] Comparative Example 5, in contrast to Example 34, the components, concentrations and preparation process of 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 polyanion 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 process of sST2 antibody-coated latex particles, the particle size of small particle size latex particles is 50-100 nm, the particle size of large particle size latex particles is 300-420 nm, the concentration of pH 5.0 MES buffer is 10 mM, the concentration of double particle size latex particles is 70 mg / L, the crosslinking agent is 12 mM EDC and 35 mM NHS, and the quenching agent is 0.05 M TCEP; after adding human sST2 antibody blocking reaction for 2 hours, the pH of the diluent of sST2 antibody-coated latex particles is adjusted to 6.5, reagent 2 stock solution is added, and 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 to 6.5 again.

[0114] Comparative Example 6, in contrast to Example 36, the components, concentrations and preparation process of 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 polyanion is 0.3 mmol / L APAM, the preservative is 0.1% sodium azide, the first surfactant is 0.05% alkyl glycoside, 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 process of sST2 antibody coated latex particles, the particle size of small particle size latex particles is 50-100 nm, the particle size of large particle size latex particles is 300-420 nm, the concentration of pH 5.0 MES buffer is 10 mM, the concentration of double particle size latex particles is 60 mg / L, the crosslinking agent is 13 mM EDC and 40 mM NHS, and after the crosslinking reaction, 0.05 M TCEP is added to quench EDC for not more than 10 minutes, then the human sST2 antibody is added for blocking reaction for 2 hours, the pH of the sST2 antibody coated latex particle diluent is adjusted to 6.5, the reagent 2 stock solution is added to make the final concentration of sST2 antibody coated latex particles in reagent 2 be 1.5 mg / mL, and the pH of reagent 2 is finally adjusted to 6.0.

[0115] Example 38, performance index evaluation of sST2 latex reagent

[0116] Determination method: the sST2 latex reagent prepared in the above examples 1-37 and comparative examples 1-4 is determined by two-point endpoint method, and the determination process is as follows: three kinds of reaction tubes are set, including blank tube, sample tube and calibration tube, wherein 10 μL purified water is added to the blank tube, 10 μL sample to be tested is added to the sample tube, and 10 μL standard is added to the calibration tube; 150 μL of reagent 1 solution is added to each of the three kinds of reaction tubes, and incubated at 37°C for 5 minutes; 50 μL of reagent 2 solution is added to each reaction tube, mixed for 80 seconds, and then the absorbance (A1) of the three kinds of reaction tubes is read by the automatic biochemical analyzer; after incubation at 37°C for 4 minutes, the absorbance (A2) of the three kinds of reaction tubes is read again by the automatic biochemical analyzer. The determination principle is the absorbance difference ΔA (i.e. the method of determining the concentration or activity of the substance to be tested by determining the total change of the product or substrate concentration from the beginning of the reaction to the equilibrium of the reaction), and the calculation formula is as follows: ΔA=A2-A1, . The reaction tube setting and sample amount are shown in the following table 1.

[0117] Table 1 Determination method of sST2 latex reagent

[0118] ;

[0119] Sensitivity: The sensitivity of the sST2 latex reagent is mainly affected by the preparation process of the sST2 antibody-coated latex particles. Take the standard sample with a concentration of 54 ng / mL as the sample to be tested, and take the sST2 latex reagents prepared by Examples 1-37, Comparative Examples 1-6 and third-party reagents, respectively, and repeat the detection of the sample tube 3 times. The detection results of the absorbance difference value of the sample tube are shown in Table 2 below. According to the absorbance difference value (ΔA sample) of the sample tube, the greater the difference, the higher the sensitivity.

[0120] Table 2 Sensitivity

[0121] ;

[0122] From the sensitivity detection results shown in Table 2, it can be seen that under the condition of ensuring the detection repeatability (smaller coefficient of variation CV% value, i.e. CV% <1%), the average difference value of the relatively larger examples is Examples 9, 11, 12, 18, 27 and 34. First, Examples 1-35 all have quenching process in the preparation process of human sST2 antibody-coated latex particles. Second, the larger the gap between the small particle size and the large particle size of the latex particles in Examples 11, 12, 27 and 34, i.e. when the latex particles with particle sizes of 50-100 nm and 300-420 nm are mixed to prepare, both the repeatability of the detection is relatively stable and the high sensitivity with relatively larger difference value can be obtained, especially the repeated detection value of Example 11 is relatively the most stable (the smallest coefficient of variation CV% is 0.13%). Further grouping and comparing the particle size of the latex particles and the type of the quenching agent shows that: compared with double particle size combination 2 (Examples 1-9 are 100-150 nm and 300-420 nm combination), double particle size combination 1 (Examples 26-33 are 50-100 nm and 300-420 nm combination) has a slightly significant effect on improving the sensitivity; compared with quenching agent group 1 (the quenching agents of Examples 34-37 are TCEP or no quenching), quenching agent group 1 (the quenching agents of Examples 26-33 are QSY-21) has a very significant difference in the positive effect on improving the sensitivity, and QSY-21 is better than TCEP, and TCEP is better than no quenching agent. It can be seen that the positive effect of the quenching agent is greater than the cooperation of the double particle size of the latex particles. It can be known from the above that whether to use quenching agent, the type of the quenching agent and the size of the double particle size of the latex particles can synergistically improve the sensitivity of the reagent.

[0123] Linear range: First, soluble growth stimulating expression gene 2 recombinant protein was added to MES buffer to prepare two concentration samples with high value (432 ng / mL) and low value (10 ng / mL), respectively. Then, the two concentration samples were mixed and diluted at different ratios to further 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, respectively. The 43 sST2 latex reagents prepared in the above 37 examples and 6 comparative examples were used to determine the six different concentration samples in parallel, each determination was repeated three times, and the average value was taken as the detection result. In addition, the sample amount of the highest point 432 ng / ml was increased by 2 times and 3 times, each determination was repeated three times, and the average value was taken as the detection result. The detection results are shown in Table 3.

[0124] Table 3 Determination results

[0125] ;

[0126] In the detection results in the range of 5 ng / mL to 432 ng / mL shown in Table 3, the correlation coefficients of the detection results of only three groups of sST2 latex reagents of Example 36, Comparative Example 1 and Comparative Example 2 are less than 0.975. That is, among the 43 sST2 latex reagents prepared in the application, 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 results of 2 times and 3 times high value before quenching can also be located on the line of the linear range. The correlation coefficients of the three sST2 latex reagents of Example 36 (r = 0.9558) without quenching, Comparative Example 1 (r = 0.8784) with ultra-low concentration buffer and Comparative Example 2 (r = 0.9674) with ultra-low concentration inorganic salt are relatively low in the range of 5 ng / mL to 432 ng / mL. There are a total of 31 sST2 latex reagents with a correlation coefficient greater than 0.99. Further comparison found that there are still 8 with a correlation coefficient greater than 0.999, which are: Example 3, Example 10, Example 11, Example 12, Example 22, Example 23, Example 29 and Example 31.

[0127] 3. Comparative analysis of group 3: 1) Among the 8 sST2 latex reagents with high correlation coefficients (r>0.999), the detection values of the high-value (432 ng / mL) custom sample were greater than 400 ng / mL in Examples 3, 11, 12, and 29. Further research on the preferred components and concentrations of these 4 examples found that: in reagent 1, the polyanion was all APAM greater than 0.3 mmol / L, the accelerator was all PVP at 0.01%, the surfactant was all alkyl glycoside greater than 0.05%, and the stabilizer was 0.1% bovine serum albumin. It is speculated that polyanions, accelerators, surfactants, and stabilizers have a greater impact on improving the linear range of reagent detection, especially on the accuracy of high-value detection. 2) The extreme minimum value (78.5 ng / mL) of the detection value of the high-value (432 ng / mL) custom sample was Example 8. Although Example 8 also included a stabilizer with similar components and concentrations as described above (Example 8 had a relatively high correlation coefficient, r=0.9908), the polyanion, first surfactant, and accelerator were all different from the above-mentioned 4 preferred examples. Therefore, it can be seen that polyanions, surfactants, and accelerators have a very important influence on improving the accuracy of high-value detection. 3) Among the 6 comparative examples, except for Comparative Example 5, the detection values of the high-value (432 ng / mL) custom sample of the other 5 comparative examples were relatively low (all ≦201.2 ng / mL). Therefore, careful study of the components of Comparative Example 5, which significantly increased the high-value detection value, found that: in Comparative Example 5, the polyanion, accelerator, and stabilizer included the above-mentioned preferred components and concentrations (r=0.994), and the biggest difference between Comparative Example 5 and the above-mentioned 4 preferred examples was that the quenching agent was TCEP during the preparation of the sST2 antibody-coated latex particles, and a very low concentration of the first surfactant (0.01% alkyl glycoside) was used in reagent 1. However, the detection value of the high-value was relatively high (356.1 ng / mL), further proving that the polyanion and accelerator, which include the same preferred components and concentrations, are of great significance to improving the accuracy of high-value detection. 4) Among the pre-zone detection results of 2-fold and 3-fold high-value concentrations, the pre-zone test values of Examples 11, 12, and 29 can even reach 3 times the linear high-value (432 ng / mL), basically reaching the level of the magnetic microparticle chemiluminescence method represented by the third-party reagent. This can further expand the detection range of high-value and improve the accuracy of high-value detection, especially the addition of BS3 as a biochemical crosslinking agent in reagent 2 of these 3 groups of examples helps to further promote the detection value to 3 times the linear high-value, and Example 11 is the most obvious, even significantly better than the third-party reagent.

[0128] From the above comparative analysis of the three groups, it can be seen that: the preferred components and concentrations of polyanion, accelerator and biochemical crosslinking agent have a greater impact on expanding the linear range of detection and improving the detection accuracy of high values, that is, the preferred polyanion is 0.3-0.6 mmol / L APAM, and the preferred accelerator is 0.01% PVP. In addition, the surfactant containing 0.05%-0.1% alkyl glycoside also has a certain degree of positive effect on improving the accuracy of high value detection, and the preferred stabilizer is 0.1% bovine serum albumin, which may have a certain positive effect on maintaining the linearity of the detection results. It can also be found in Table 3 that if the error of high value (432 ng / mL) is to be ensured to be less than 5% (95-105%) within the confidence interval, the detection value should be between 410 ng / mL and 453 ng / mL, and among the 37 examples and 6 comparative examples, only examples 11, 12 and 29 can meet this requirement. It can be seen that in the case of clinical common sST2 concentration up to 400 ng / mL, the detection accuracy of the high value part still needs to be optimized and solved, and at least in order to solve the technical problem of high value detection of sST2, polyanion and accelerator are a key component.

[0129] In Table 3, the highest value of the correlation coefficient (r) is example 11 (r=0.9999), and based on the detection data of example 11, the theoretical concentration (xi) in the range of 5 ng / mL-432 ng / mL is taken as the independent variable, and the average value of the measured results (yi) of example 11 is taken as the dependent variable. The linear evaluation result graph of example 11 is drawn as shown in Figure 1 The regression equation corresponding to example 11 is Y=0.9635x-1.6071 (R 2 =0.9998), it can be seen that there is a good linear relationship between the measured value and the theoretical value of example 11, and the determination coefficient (R 2 ) is very close to 1, indicating that within the linear range of 5 ng / mL-432 ng / mL, the detection value of example 11 is more reliable, the fitting degree is extremely high, and has a good linear relationship.

[0130] Anti-interference ability: The sST2 latex reagents prepared in Examples 1-37 and Comparative Examples 1-6 were selected as the anti-interference ability evaluation objects, and third-party reagents were used as controls. The samples to be tested were from the same large amount of human serum sample, and the serum sample was used as the matrix. Rheumatoid factor RF, hemoglobin HB, and triglyceride TG were added to prepare simulated samples to be tested. The above sST2 latex reagents were used to perform parallel comparison on the high-value interference simulated samples to be tested, each determination was repeated 3 times, the mean value was calculated, and the relative deviation was calculated. The addition amount of rheumatoid factor RF, hemoglobin HB, and triglyceride TG was 0, which was used as the negative control for interference detection. The determination results of the three types of interference simulated samples (referred to as interference samples) are shown in Table 4.

[0131] Table 4 Anti-interference ability of interference substances

[0132]

[0133] Detailed analysis of the detection results of the high-value interfering samples of the common interfering substances (rheumatoid factor RF, hemoglobin HB, and triglyceride TG) in the three types of serum samples shown in Table 4 can find that: only the relative deviations of the detection values of the three types of interfering samples of Example 11, Example 15, Example 16, Example 29, Comparative Example 2, and the third-party reagent are less than 10%, and the average values of the relative deviations are 2.75%, 5.12%, 6.02%, 6.86%, 5.77%, and 7.13%, respectively. It can be seen that the anti-interference performance of Example 11 is not only significantly better than that of other preferred examples and comparative examples, but also significantly better than that of the magnetic microparticle chemiluminescence method represented by the third-party reagent. First, the components and preparation processes found by comparing the five groups of Example 11, Example 15, Example 16, Example 29, and Comparative Example 2 are as follows: the preparation process of the human sST2 antibody-coated latex particles, the pairing of the latex particles (50-100 & 300-420), and the quencher (QSY-21) are all the same, the components and concentrations of the buffer, stabilizer, and promoter are the same, and the pH values of Reagent 1 are all 6.5. It is preliminarily inferred that the synergistic effect of human sST2 antibody-coated latex particles, buffer, stabilizer, promoter, and pH value has a significant influence on improving the anti-interference ability of the reagent; second, the anti-interference performance of Example 20 (the average value of the relative deviation is the largest, which is 17.64%) is significantly worse than that of other examples or comparative examples, which can be regarded as the worst combination of anti-interference formula components and concentrations. By comparing Example 20 with the above-mentioned three groups of anti-interference ability preferred examples, it is found again that the pairing of double-particle-size latex particles (50-100+300-420 & 50-100+200-300), the buffer (HEPES+MES & phosphate), the stabilizer (BSA & D-mannitol), the promoter (polyvinylpyrrolidone & PEG6000), and the pH value of Reagent 1 (6.5 & 7) are significantly different. The anti-interference ability of Example 20, which is poor, reversely verifies that the synergistic effect of the pairing of double-particle-size latex particles in the preferred human sST2 antibody-coated latex particles, the buffer, the stabilizer, the promoter, and the pH value of Reagent 1 has a significant influence on improving the anti-interference ability of the reagent; third, among the above-mentioned five groups of anti-interference ability preferred examples, the relative deviations of all detection values of the three types of interfering samples of Example 11 are less than 5% and the average value of the relative deviations is significantly the smallest (2.75%), which means that the anti-interference ability of the sST2 latex reagent prepared by Example 11 is significantly better than that of other examples or comparative examples. It can be regarded as the optimal combination of formula components and concentrations, i.e., the sST2 latex reagent prepared by Example 11 has relatively strong anti-interference ability for clinical samples containing high concentrations of rheumatoid factor (600 IU / mL), hemoglobin (800 IU / mL), and triglyceride (1.5%), and the accuracy of the measured value is relatively reliable.

[0134] From the above analysis, it can be seen that the preferred components, concentrations and preparation process of the sST2 latex reagent, at least the above-mentioned preferred conditions of double-particle-size latex particle pairing, buffer, stabilizer, accelerator and pH value, can significantly improve the interference ability of the reagent.

[0135] Stability: After placing 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, the same sample to be tested was measured to evaluate the stability of the sST2 latex reagent, wherein the sample to be tested was derived from the same serum sample, which was stored at -80°C after being aliquoted, and was randomly taken out for serum sST2 determination experiment at the time of measurement, and each determination was repeated 3 times. The linear change of the actual measured mean value of the stability test with storage time is shown in Figure 2 Firstly, the actual measured value of the sST2 latex reagent prepared by Example 11 placed at 2-8°C for 0, 1, 3, 6, 12, 18 and 24 months changes relatively little with storage time, and the stability is relatively best, at least the effective period of storage at 2-8°C can be as long as 24 months, and the actual measured mean value of the third-party reagent also changes little with storage time, but the actual measured value after storage at 2-8°C is slightly higher than that before low-temperature storage. Secondly, Figure 2 Further comparison found that compared with Example 11, the stability of the actual measured value corresponding to Comparative Examples 1, 3, 4 and 5 all showed extremely significant differences with the increase of storage time. Further analysis of the components of different sST2 latex reagents showed that the amount of buffer in Reagent 1 and Reagent 2 of Comparative Example 1 was as low as 15 mmol / L, Comparative Example 3 used a very small amount of second stabilizer in Reagent 2, Comparative Example 4 added a very small amount of polyanion and a very small amount of second surfactant in Reagent 2, and Comparative Example 5 used a very small amount of first surfactant in Reagent 1. It can be seen that appropriate concentrations of buffer, second stabilizer, polyanion and surfactant have a significant impact on maintaining the low-temperature storage stability of sST2 latex reagent. Thirdly, Figure 2 It can be seen that the actual measured value corresponding to Comparative Example 1 showed a very significant decrease with the increase of storage time, which can be speculated that appropriate buffer concentration has a very significant effect on maintaining the buffer environment of antigen-antibody specific binding reaction; relatively speaking, Figure 2It can also be seen that the measured values corresponding to Comparative Example 3 increase significantly with the increase of storage time, which can be deduced that the appropriate concentration of the second stabilizer in Test Agent 2 has a significant effect on avoiding non-specific self-aggregation of antibody-coated latex particles. Fourthly, the significant difference in the measured values corresponding to Comparative Example 4 and Comparative Example 5 further proves that polyanions and surfactants also have a significant effect on maintaining the stability of the latex reagent. Fifthly, compared with Example 11, the measured values of Examples 18-33 also show significant changes with the increase of storage time, which further verifies that the preferred components and concentrations of the buffer, stabilizer, polyanion and surfactant have important effects on maintaining the low-temperature storage stability of the sST2 latex reagent. Finally, the measured values corresponding to Example 11 in the stability evaluation results do not show significant fluctuations with the increase of storage time, and the stability is optimal. It can be seen that the components and proportions of Example 11 are relatively optimal, and the stability evaluation further verifies the positive effects of the preferred components and concentrations of the buffer, stabilizer, polyanion and surfactant in Example 11 on maintaining the stability of the reagent, such as the first buffer preferably 50 mmol / L HEPES buffer, the second buffer preferably 50 mmol / L MES buffer, the first stabilizer and the second stabilizer both preferably 0.1% bovine serum albumin, the polyanion preferably 0.3 mmol / L APAM, and the first surfactant and the second surfactant both preferably 0.05% alkyl glycoside. The stability experimental results of the sST2 latex reagent are relatively optimal. Based on the above analysis, it can be seen that the stability of the sST2 latex reagent is affected by multiple factors, mainly including buffer, stabilizer, polyanion and surfactant and other factors.

[0136] Repeatability: The test sample was a randomly selected same large dose of clinical serum sample, and the sST2 latex reagents prepared by 37 examples and 6 comparative examples of the present application and third-party reagents were used to determine the aliquoted test sample, each determination was repeated 10 times, the repeatability of the sST2 latex reagent was evaluated by calculating the average value and coefficient of variation (CV) of the measured values, and the repeatability determination results are shown in Table 5.

[0137] Table 5 Repeatability determination results

[0138]

[0139] Generally, the coefficient of variation (CV) less than 10% is considered to be an acceptable range of repeatability, which is equivalent to good repeatability of reagent detection. As can be seen from Table 5, the differences between the average values of the measured values of 37 examples and 6 comparative examples are not large (all less than 10%), and the coefficient of variation of 12 samples is less than or equal to 2.5%, especially the coefficient of variation between the repeated detection results of Example 11 (0.3 mmol / l APAM) is the smallest (0.7%), which is the only representative of the coefficient of variation (CV) less than 1%, and it can be seen that the repeatability of the sST2 latex reagent prepared by Example 11 has a very significant beneficial effect.

[0140] Correlation: According to the above performance verification results, the evaluation results of the sensitivity, linear range, low temperature storage stability and repeatability of the sST2 latex reagent prepared by Example 11 of the present application are relatively better, so the sST2 latex reagent prepared by Example 11 and the third party reagent are used to detect the same sample to be tested in parallel to evaluate the correlation of Example 11, wherein the sample to be tested is 60 randomly selected clinical serum samples, and the measured values of the same sample to be tested by the two kinds of reagents are used as the horizontal and vertical coordinates, respectively, to draw the correlation curve of the measured values of the two kinds of reagents, as shown in Figure 3 The regression equation is y = 1.0021x - 0.5823 (R 2 = 0.9938), it can be seen that the determination results of the two kinds of reagents are highly consistent, showing good linear correlation, that is, the measured value of the sST2 latex reagent prepared by Example 11 is good, and has good accuracy and correlation.

[0141] The performance indicators of the sST2 latex reagents prepared in 37 examples and 6 comparative examples can be seen: the application optimizes the preferred components and concentrations of buffers, stabilizers, polyanions, surfactants and accelerators in reagent 1 and reagent 2, and the performance evaluation results prove that: whether the quenching agent is used in the preparation process of sST2 antibody coated latex particles, the type of quenching agent and the combination of double particle size latex particles have a synergistic effect and a positive influence on improving the sensitivity of sST2 latex reagent; the preferred components and concentrations of polyanions and biochemical cross-linking agents have a positive influence on expanding the linear range of detection and improving the accuracy of high value detection; due to the diversity of interfering substances, the application at least proves that the preferred conditions of double particle size latex particle pairing, buffer, stabilizer, accelerator and pH value have a synergistic effect and a positive influence on improving the interference ability of the reagent; the preferred conditions of buffer, stabilizer, polyanion, biochemical cross-linking agent and surfactant have a synergistic effect and a positive influence on improving the stability of the reagent. In addition, the repetitive detection data corresponding to the 37 examples and 6 comparative examples of the application all meet the requirements. In addition, the pre-experiment of the application found that when using latex enhanced immunoturbidimetry to determine soluble growth stimulating expression gene 2 protein, higher concentration of inorganic salt is not conducive to improving the detection sensitivity and repeatability, so the inorganic salt concentration is greatly reduced to 0.1%-0.2%, which greatly improves the comprehensive performance of the reagent under the synergistic effect of the optimization of other key components, especially the sensitivity, anti-interference ability, stability, repeatability of the sST2 latex reagent prepared in example 11, and the correlation with the third party reagent detection is relatively better, and the existing common detection methods of soluble growth stimulating expression gene 2 (sST2) protein cannot meet the detection needs of high concentration sST2 greater than 400 ng / mL, especially the latex enhanced immunoturbidimetry for detecting sST2 has the phenomenon that the high value quantitative detection value cannot be increased, and the above shows that the preferred components and concentrations of polyanions, biochemical cross-linking agents and accelerators creatively added in example 11 of the application have more key significance for expanding the linear detection range and improving the accuracy of high value detection, and the evaluation results of the comprehensive performance indicators not only reach and are better than the third party reagent, which shows that the performance of the preferred latex reagent of the application can reach the level of magnetic microparticle chemiluminescence method, and is particularly suitable for monitoring or evaluating the severity of heart failure under high sST2 concentration.

[0142] The specific embodiments of the application have been described in detail, so that those skilled in the art will easily understand. However, different or similar modifications or replacements can be made to some specific details according to all the descriptions disclosed, and these changes are within the protection scope of the application. The whole scope of the application 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 an organic compound as a quencher, wherein the quencher is either QSY-21 or TCEP. The biochemical crosslinking agent is sodium bis(succinimide) octanoate (BS3).

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%.

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 resistant to soluble growth-stimulating gene 2, 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 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, second stabilizer, preservative, biochemical cross-linking agent, and second surfactant to the second buffer solution in sequence according to the predetermined components and concentrations, and mix well. Adjust the pH value to 6 to complete the preparation of the reagent 2 stock solution. Add the reagent 2 stock solution to the preparation diluent of soluble growth-stimulating gene 2 protein antibody-coated latex particles, 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.

8. The application of 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, and the preparation method of the soluble growth-stimulating gene 2 protein assay kit according to claim 7 in the preparation of latex reagent-related products.

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