阅读说明：本技术 一种脂联素-胶乳增强免疫比浊试剂盒及其制备方法 (Adiponectin-latex enhanced immunoturbidimetry kit and preparation method thereof ) 是由 袁嘉扬 单以朗 于 2019-09-12 设计创作，主要内容包括：本发明涉及生物技术领域,涉及一种脂联素-胶乳增强免疫比浊试剂盒的制备方法,公开了试剂R2由反应液、保存液以及胶乳微球抗体偶联物组成,其反应液由第二缓冲液构成,胶乳微球抗体偶联物由小胶乳微球、大胶乳微球、生物素、链霉亲和素、脂联素单抗、脂联素多抗以及1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐构成。优点是：经多次研发实验后,我司采用大小胶乳微球方案：大胶乳联脂联素单抗增加低端灵敏度；小胶乳连接链霉亲和素按比例混合,脂联素多抗连接生物素按比例混合,二者再按比例混合反应,可提高线性范围；将两种胶乳按照比例混合,可改善重复性和灵敏度线性范围可做到1.00-32.00mg/L。(the invention relates to the technical field of biology, and relates to a preparation method of an adiponectin-latex enhanced immunoturbidimetric kit, and discloses a reagent R2 which consists of a reaction solution, a preservation solution and a latex microsphere antibody conjugate, wherein the reaction solution consists of a second buffer solution, and the latex microsphere antibody conjugate consists of small latex microspheres, large latex microspheres, biotin, streptavidin, adiponectin monoclonal antibodies, adiponectin polyclonal antibodies and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. The advantages are that: after many research and development experiments, I have adopted the scheme of the large and small latex microspheres: the large latex adiponectin monoclonal antibody increases the low-end sensitivity; the small latex is connected with the streptavidin and mixed in proportion, the adiponectin and the polyclonal antibody are connected with the biotin and mixed in proportion, and the two are mixed in proportion for reaction, so that the linear range can be improved; the two latexes are mixed according to a proportion, so that the repeatability and the sensitivity can be improved, and the linear range can be 1.00-32.00 mg/L.)

1. An adiponectin-latex enhanced immunoturbidimetry kit, which is characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein each component and concentration of the reagent R1 comprises:

the reagent R2 is composed of reaction liquid, preservation liquid and a latex microsphere antibody conjugate, wherein the reaction liquid is composed of a second buffer solution, the preservation liquid is composed of glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate is composed of small latex microspheres, large latex microspheres, biotin, streptavidin, adiponectin monoclonal antibody, adiponectin polyclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows:

2. the adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the first buffer solution in the reagent R1 comprises one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.

3. The adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the second buffer solution in the reagent R2 comprises one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution, Tris buffer solution and the like.

4. the adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the first stabilizer and the second stabilizer in the reagent R1 and the reagent R2 both comprise one or more of casein, mannitol and bovine serum albumin.

5. The adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the surfactant in the reagent R1 comprises one or more of Tween 20, Triton X-100, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.

6. the adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the first preservative in the reagent R1 and the second preservative in the reagent R2 comprise one of sodium azide, Proclin-950, Proclin-300 and thimerosal.

7. The adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the small latex microspheres in the reagent R2 have the selected particle size of 15-85nm, and the large latex microspheres have the selected particle size of 270-630 nm.

8. The adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the adiponectin monoclonal antibody in the reagent R2 is a mouse monoclonal antibody; the adiponectin polyclonal antibody is one of sheep polyclonal antibody and rabbit polyclonal antibody.

9. the adiponectin-latex-enhanced immunoturbidimetry kit according to claim 1, wherein: the pH of the R1 reagent is between 5.90 and 8.90; the pH of the R2 reagent is between 6.00 and 8.00.

10. a preparation method of an adiponectin-latex enhanced immunoturbidimetry kit is characterized by comprising the following steps:

the method comprises the following steps: A) reagent R1 preparation:

adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding a first buffer solution, sodium chloride, a first stabilizer, a surfactant, polyethylene glycol 6000 and a first preservative while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 5.90-8.90 and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

B) reagent R2 preparation:

Preparation of B1 reaction solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding a second buffer solution while stirring until the materials are completely dissolved, keeping the materials clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, adjusting the pH value to 5.50-6.50, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding glycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-8.00, continuously stirring for 5-10 minutes until all the materials are completely dissolved, enabling the solution to be clear and transparent, enabling the bottom of the liquid preparation tank to have no precipitate, and fixing the volume to the final volume;

Filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;

b3 preparation of latex microsphere antibody conjugate:

The method comprises the following steps: diluting the small latex microspheres to the concentration of 0.05-2.0% by using reaction liquid, dialyzing the small latex microspheres and streptavidin according to the ratio of 1:2-1:5 for overnight combination;

diluting adiponectin polyclonal antibody to 0.05-3.0g/L concentration with reaction solution, dialyzing with biotin at ratio of 1:1-1:3 overnight for combination;

mixing the latex-streptavidin and biotin-adiponectin polyclonal antibody according to the ratio of 0.5:1-4: 1;

dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.005-0.3g/L, dripping the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding bovine serum albumin, sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the last time to remove the supernatant, adding the preservation solution, and ultrasonically resuspending;

step two: diluting the large latex microspheres to the concentration of 0.05-2.0% by using reaction liquid, diluting the adiponectin monoclonal antibody to the concentration of 0.05-3.0g/L by using the reaction liquid, and uniformly mixing the two according to the proportion of 0.5:1-4: 1;

dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.005-0.3g/L, dripping the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding bovine serum albumin, sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the last time to remove the supernatant, adding the preservation solution, and ultrasonically resuspending;

step three: and (3) uniformly mixing the latex successfully coupled in the step one and the latex successfully coupled in the step two according to the ratio of 2:1-5:1, filling the mixture into a finished product tank, and metering to a final concentration, wherein the obtained product is marked as R2.

Technical Field

the invention relates to the technical field of biology, relates to an adiponectin-latex enhanced turbidimetric immunoassay kit, and particularly relates to a preparation method of the adiponectin-latex enhanced turbidimetric immunoassay kit.

background

adiponectin (APN), also known as Acrp30, GBP28 or AdipoQ, is a cytokine that is synthesized and secreted by adipocytes and is stably present in human plasma at a concentration ranging from about 3.0 to 30.0mg/L, which accounts for about 0.01% of plasma protein. Human adiponectin is 244 amino acids and includes 3 regions: an amino-terminal signal sequence, a collagen domain, and a carboxy-terminal globular domain. Adiponectin exists in blood as three subtypes, trimer (65kb), hexamer (150kb), and high molecular weight polymer (18-36 multimers, >280 kb). Clinical studies show that adiponectin is closely related to obesity, type II diabetes, coronary heart disease, and insulin resistance, and has anti-atherogenesis, anti-inflammation, and anti-intimal hyperplasia properties after vascular injury.

Adiponectin is measured by various methods, such as Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) (human adiponectin ELISA kit, CN 102517256A). Although the ELISA method is clinically used for nearly twenty years, it still has some fatal disadvantages, long operation time and low automation degree. Radioimmunoassay (RIA) is highly sensitive, but is unstable, less reproducible than ELISA, and poses the risk of radioactive contamination.

Disclosure of Invention

The purpose of the invention is: aiming at the defects, the adiponectin-latex enhanced immunoturbidimetry kit and the preparation method thereof are provided.

in order to achieve the purpose, the invention adopts the technical scheme that:

an adiponectin-latex enhanced immunoturbidimetry kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration:

the reagent R2 is composed of reaction liquid, preservation liquid and a latex microsphere antibody conjugate, wherein the reaction liquid is composed of a second buffer solution, the preservation liquid is composed of glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate is composed of small latex microspheres, large latex microspheres, biotin, streptavidin, adiponectin monoclonal antibody, adiponectin polyclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows:

0.005-0.3g/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

The first buffer solution in the reagent R1 comprises one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.

the second buffer solution in the reagent R2 comprises one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution, Tris buffer solution and the like.

the first stabilizer and the second stabilizer in the reagent R1 and the reagent R2 both comprise one or more of casein, mannitol and bovine serum albumin.

The surfactant in the reagent R1 comprises one or more of Tween 20, Triton X-100, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.

the first preservative in the reagent R1 and the second preservative in the reagent R2 comprise one of sodium azide, Proclin-950, Proclin-300 and thimerosal.

the small latex microspheres in the reagent R2 have the selected particle size of 15-85nm, and the large latex microspheres have the selected particle size of 270-630 nm.

The adiponectin monoclonal antibody in the reagent R2 is a mouse monoclonal antibody; the adiponectin polyclonal antibody is one of sheep polyclonal antibody and rabbit polyclonal antibody.

the pH of the R1 reagent is between 5.90 and 8.90; the pH of the R2 reagent is between 6.00 and 8.00.

a preparation method of an adiponectin-latex enhanced immunoturbidimetry kit is characterized by comprising the following steps: the method comprises the following steps: A) reagent R1 preparation:

adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding a first buffer solution, sodium chloride, a first stabilizer, a surfactant, polyethylene glycol 6000 and a first preservative while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 5.90-8.90 and fixing the volume to the final volume;

Filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

B) Reagent R2 preparation:

Preparation of B1 reaction solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding a second buffer solution while stirring until the materials are completely dissolved, keeping the materials clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, adjusting the pH value to 5.50-6.50, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding glycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-8.00, continuously stirring for 5-10 minutes until all the materials are completely dissolved, enabling the solution to be clear and transparent, enabling the bottom of the liquid preparation tank to have no precipitate, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;

B3 preparation of latex microsphere antibody conjugate:

the method comprises the following steps: diluting the small latex microspheres to the concentration of 0.05-2.0% by using reaction liquid, dialyzing the small latex microspheres and streptavidin according to the ratio of 1:2-1:5 for overnight combination;

Diluting adiponectin polyclonal antibody to 0.05-3.0g/L concentration with reaction solution, dialyzing with biotin at ratio of 1:1-1:3 overnight for combination;

mixing the latex-streptavidin and biotin-adiponectin polyclonal antibody according to the ratio of 0.5:1-4: 1;

Dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.005-0.3g/L, dripping the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding bovine serum albumin, sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the last time to remove the supernatant, adding the preservation solution, and ultrasonically resuspending;

Step two: diluting the large latex microspheres to the concentration of 0.05-2.0% by using reaction liquid, diluting the adiponectin monoclonal antibody to the concentration of 0.05-3.0g/L by using the reaction liquid, and uniformly mixing the two according to the proportion of 0.5:1-4: 1;

dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.005-0.3g/L, dripping the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding bovine serum albumin, sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the last time to remove the supernatant, adding the preservation solution, and ultrasonically resuspending;

Step three: and (3) uniformly mixing the latex successfully coupled in the step one and the latex successfully coupled in the step two according to the ratio of 2:1-5:1, filling the mixture into a finished product tank, and metering to a final concentration, wherein the obtained product is marked as R2.

Compared with the prior art, the invention achieves the technical effects that: after many research and development experiments, I have adopted the scheme of the large and small latex microspheres: the large latex adiponectin monoclonal antibody increases the low-end sensitivity; the small latex is connected with the streptavidin and mixed in proportion, the adiponectin and the polyclonal antibody are connected with the biotin and mixed in proportion, and the two are mixed in proportion for reaction, so that the linear range can be improved; the two latexes are mixed according to a proportion, so that the repeatability and the sensitivity can be improved, and the linear range can be 1.00-32.00 mg/L.

drawings

FIG. 1 is a calibration graph of example 4 of the present invention.

Detailed Description

an adiponectin-latex enhanced immunoturbidimetry kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration: 6.0-12.2g/L of first buffer solution, 0.5-2g/L of first stabilizer, 5.0-27.0g/L of sodium chloride, 1-5ml/L of surfactant, 600060-120g/L of polyethylene glycol, 0.8-2.0ml/L of first preservative;

the reagent R2 is composed of reaction liquid, preservation liquid and a latex microsphere antibody conjugate, wherein the reaction liquid is composed of a second buffer solution, the preservation liquid is composed of glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate is composed of small latex microspheres, large latex microspheres, biotin, streptavidin, adiponectin monoclonal antibody, adiponectin polyclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows: 8.05-27.30g/L of second buffer solution, glycine, 1.26-9.83g/L of second stabilizer, 0.5-2.0g/L of second preservative, 0.8-2.0ml/L of small latex microspheres, 0.05-2.0% of large latex microspheres, 0.05-2.0% of biotin, 5-20g/L of streptavidin, 5-20g/L of adiponectin monoclonal antibody, 0.05-3.0g/L of adiponectin polyclonal antibody, 0.05-3.0g/L of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and 0.005-0.3g/L of adiponectin polyclonal antibody.

the first buffer solution in the reagent R1 includes one or more of PBS buffer solution, HEPES buffer solution, MES buffer solution, and Tris buffer solution, in an embodiment, the preferred first buffer solution is preferably Tris, and the concentration thereof may preferably be: 6.0g/L, 9g/L and 12.2 g/L.

the second buffer solution in the reagent R2 comprises one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution, Tris buffer solution and the like, in the embodiment, the preferred second buffer solution is MES (2- (N-morpholine) ethanesulfonic acid monohydrate, and the concentration thereof can be preferably 8.05g/L, 18g/L and 27.3 g/L.

The first stabilizer and the second stabilizer in the reagents R1 and R2 each include one or more of casein, mannitol, and bovine serum albumin, and in the embodiment, the first stabilizer in the reagent R1 is preferably: casein, mannitol, bovine serum albumin, corresponding concentrations are: 0.5g/L, 1.2g/L and 2 g/L; the second stabilizer in the reagent R2 is preferably: casein, mannitol, bovine serum albumin, corresponding concentrations are: 0.5g/L, 1.2g/L and 2 g/L.

the surfactant in the reagent R1 comprises one or more of Tween 20, Triton X-100, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether, and in the embodiment, the surfactant in the reagent R1 is preferably: tween 20, Triton X-100 and polyvinylpyrrolidone, wherein the corresponding concentrations are as follows: 1ml/L, 3ml/L, 5 ml/L.

the first preservative in the reagent R1 and the second preservative in the reagent R2 comprise one of sodium azide, Proclin-950, Proclin-300 and thimerosal, and in the embodiment, the first preservative in the reagent R1 is preferably: sodium azide, Proclin-950 and Proclin-300, wherein the corresponding concentrations are respectively as follows: 0.8ml/L, 1.4ml/L and 2 ml/L; in embodiments, the second preservative in the agent R2 is preferably: sodium azide, Proclin-950 and Proclin-300, wherein the corresponding concentrations are respectively as follows: 0.8ml/L, 1.4ml/L and 2 ml/L.

the selected particle size of the small latex microspheres in the reagent R2 is 15-85nm, the selected particle size of the large latex microspheres is 270-630nm, and the preferred particle sizes of the small latex microspheres in the embodiment are 12nm, 47nm and 85nm respectively; the particle sizes of the large latex microspheres are 270nm, 450nm and 630nm respectively.

the adiponectin monoclonal antibody in the reagent R2 is a mouse monoclonal antibody; the adiponectin polyclonal antibody is one of sheep polyclonal antibody and rabbit polyclonal antibody.

the pH of the R1 reagent is between 5.90 and 8.90; the pH of the R2 reagent is between 6.00 and 8.00.

a preparation method of an adiponectin-latex enhanced immunoturbidimetry kit is characterized by comprising the following steps: the method comprises the following steps: A) reagent R1 preparation:

Adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding a first buffer solution, sodium chloride, a first stabilizer, a surfactant, polyethylene glycol 6000 and a first preservative while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 5.90-8.90 and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

B) Reagent R2 preparation:

Preparation of B1 reaction solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding a second buffer solution while stirring until the materials are completely dissolved, keeping the materials clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, adjusting the pH value to 5.50-6.50, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding glycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-8.00, continuously stirring for 5-10 minutes until all the materials are completely dissolved, enabling the solution to be clear and transparent, enabling the bottom of the liquid preparation tank to have no precipitate, and fixing the volume to the final volume;

Filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;

b3 preparation of latex microsphere antibody conjugate:

The method comprises the following steps: diluting the small latex microspheres to the concentration of 0.05-2.0% by using reaction liquid, dialyzing the small latex microspheres and streptavidin according to the ratio of 1:2-1:5 for overnight combination;

Diluting adiponectin polyclonal antibody to 0.05-3.0g/L concentration with reaction solution, dialyzing with biotin at ratio of 1:1-1:3 overnight for combination;

Mixing the latex-streptavidin and biotin-adiponectin polyclonal antibody according to the ratio of 0.5:1-4: 1;

dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.005-0.3g/L, dripping the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding bovine serum albumin, sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the last time to remove the supernatant, adding the preservation solution, and ultrasonically resuspending;

Step two: diluting the large latex microspheres to the concentration of 0.05-2.0% by using reaction liquid, diluting the adiponectin monoclonal antibody to the concentration of 0.05-3.0g/L by using the reaction liquid, and uniformly mixing the two according to the proportion of 0.5:1-4: 1;

dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.005-0.3g/L, dripping the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding bovine serum albumin, sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, sucking a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the last time to remove the supernatant, adding the preservation solution, and ultrasonically resuspending;

Step three: and (3) uniformly mixing the latex successfully coupled in the step one and the latex successfully coupled in the step two according to the ratio of 2:1-5:1, filling the mixture into a finished product tank, and metering to a final concentration, wherein the obtained product is marked as R2.

the invention is further described with reference to the following figures and examples: