CN115097141A

CN115097141A - Adiponectin kit and preparation method thereof

Info

Publication number: CN115097141A
Application number: CN202210701788.3A
Authority: CN
Inventors: 李名星; 张设熙; 韦佳志; 黄多全; 何彩玉; 余思柳
Original assignee: Guangxi Kangbailai Technology Co ltd
Current assignee: Guangxi Kangbailai Technology Co ltd
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2022-09-23
Anticipated expiration: 2042-06-21
Also published as: CN115097141B

Abstract

The invention relates to an adiponectin kit and a preparation method thereof, wherein the adiponectin kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises: 25-100 mM of buffer solution A, 3-11 g/L of stabilizer A, 2-4 g/L of preservative A, 2-7 g/L of reaction enhancer and 2-5 g/L of surfactant; the R2 reagent includes: 25-100 mM of buffer solution B, 3-11 g/L of stabilizer B, 2-4 g/L of preservative B and 1-2 mg/L of latex microspheres coupled with adiponectin antibodies. The adiponectin kit has the advantages of high sensitivity, good stability, high accuracy, wide linear range, long shelf life of more than 18 months, simple and convenient operation, and can be used for clinical detection and prevention and treatment of obesity, diabetes, coronary heart disease, cardiovascular diseases and other diseases.

Description

Adiponectin kit and preparation method thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to an adiponectin kit and a preparation method and application thereof.

Background

Adiponectin (Adiponectin/ADPN) is an endogenous, biologically active polypeptide or protein secreted by adipocytes. The research on human bodies finds that the adiponectin level can indicate the development of II type diabetes and coronary heart disease, and shows the potential of resisting diabetes, atherosclerosis and inflammation in clinical tests.

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, trimers, hexamers, and high molecular weight polymers. Clinical studies show that adiponectin is closely related to type II diabetes, coronary heart disease, and insulin resistance, and has anti-atherogenesis, anti-inflammation, and anti-intimal hyperplasia properties after vascular injury. According to the data of the international diabetes association, there are currently over 3 hundred and 8 million diabetics worldwide, and 46% of them are not discovered until after serious diabetic complications occur. The adiponectin kit can detect the adiponectin level in human blood, provide valuable information for diseases such as diabetes and the like, and realize early discovery and early treatment of diabetes, thereby reducing the incidence of diabetes.

At present, the methods for detecting adiponectin mainly include chemiluminescence, enzyme-linked immunoassay, radioimmunoassay and latex immunoturbidimetry. Chemiluminescence methods, while sensitive and simple, are poorly selective and can respond to a range of compounds, rather than to a single compound, with poor specificity. The enzyme-linked immunoassay method has high sensitivity, but has complex operation and is difficult to realize automatic detection. The radioimmunoassay has the advantages of higher sensitivity, but has the defects of complicated operation, long time consumption, easy interference of manual operation and external factors, low automation degree and the like, and radioactive markers can cause harm to operators and cause environmental pollution. The latex enhanced immunoturbidimetry is widely applied by people due to the advantages of rapidness, simplicity, high precision, easy automation, suitability for simultaneous detection of large-batch specimens and the like. However, the existing latex-enhanced immunoturbidimetry detection kit still has some defects to be improved, and if the sensitivity of the kit is insufficient, the stability of the kit is insufficient, or the production efficiency is low, and the cost is high.

In order to promote the popularization and application of the adiponectin detection method, it is necessary to research an adiponectin kit with high accuracy, high sensitivity and good stability.

Disclosure of Invention

In order to solve the problems, the invention provides an adiponectin kit and a preparation method thereof, and the adiponectin kit has the advantages of high accuracy, high sensitivity, good stability, high production efficiency and the like.

In order to achieve the purpose, the scheme provided by the invention is as follows:

an adiponectin kit comprises an R1 reagent and an R2 reagent, wherein the solvent is purified water; wherein the content of the first and second substances,

the R1 reagent comprises: buffer solution A25-100 mM, stabilizer A3-11 g/L, preservative A2-4 g/L, reaction enhancer 2-7 g/L, and surfactant 2-5 g/L;

the R2 reagent comprises: 25-100 mM of buffer solution B, 3-11 g/L of stabilizer B, 2-4 g/L of preservative B and 1-2 mg/L of latex microspheres coupled with adiponectin antibodies.

Specifically, the buffer solution A and the buffer solution B are combined by two or three of glycine buffer solution, MES buffer solution and MOPS buffer solution.

Preferably, the buffer solution A and the buffer solution B are 10-50 mM of glycine buffer solution, 5-25 mM of MES buffer solution and 10-25 mM of MOPS buffer solution.

Preferably, the components of the stabilizing agent A and the stabilizing agent B are as follows: 1-3 g/L bovine serum albumin, 1-3 g/L casein and 1-5 g/L mannitol.

Preferably, the preservative A and the preservative B comprise the following components: 1-2 g/L, Proclin 3001-2 g/L sodium azide.

Preferably, the reaction enhancer component in the R1 reagent is: polyethylene glycol 60001-4 g/L and polyethylene glycol 80001-3 g/L.

Preferably, the surfactant component in the R1 reagent is: tween 201-2.5 g/L, Triton-X1001-2.5 g/L.

Preferably, the adiponectin antibody is a human adiponectin monoclonal antibody, and the addition amount of the antibody is 0.1-0.5 mg/L.

Preferably, the particle size of the latex microsphere coupled with the adiponectin antibody is 240 nm.

Specifically, the preparation method of the adiponectin kit comprises the following steps:

1) preparation of R1 reagent: according to the component content of the adiponectin R1 reagent, mixing the component substances in the same container, and uniformly mixing to obtain the adiponectin R1 reagent;

2) preparation of R2 reagent:

activating: taking 40-60% of the total amount of the buffer solution B, adding the latex microspheres, uniformly mixing by stirring in a shaking table at 37 ℃ for 10-15min, adding the coupling agent, and continuously stirring for 30-35min to uniformly mix to respectively obtain activated latex microspheres;

marking: adding the adiponectin antibody into the activated latex microspheres while stirring, and uniformly mixing the activated latex microspheres and the adiponectin antibody for 60-120min by shaking at 37 ℃ to obtain latex microspheres coupled with the adiponectin antibody;

sealing: adding a preservative B and a stabilizer B into the latex microspheres coupled with the adiponectin antibodies, fully dissolving and mixing for 40-60min, and sealing to obtain sealed latex microspheres;

adding the residual buffer solution B into the closed latex microspheres, and uniformly mixing to obtain an R2 reagent;

3) and (3) independently subpackaging the R1 reagent and the R2 reagent, and sealing and storing to obtain the adiponectin kit.

Specifically, the coupling agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and the addition amount of the coupling agent is 0.1-0.2 g/L. The latex microspheres can be activated by adding the coupling agent, so that the efficiency of the European Union reaction is improved.

The adiponectin monoclonal antibody used in the invention is purchased from Nanjing Liding Biotechnology Co., Ltd in vitro, and the latex microsphere is purchased from Japanese JSR company with particle size of 240 nm.

The invention has the following beneficial effects:

1. the adiponectin kit disclosed by the invention is high in sensitivity, good in stability, high in accuracy, wide in linear range, simple and convenient to operate, and capable of being stored for more than 18 months at the temperature of 2-8 ℃, can be used for clinical detection, and has positive significance for preventing and treating obesity, diabetes, coronary heart disease, cardiovascular diseases and other diseases.

2. The buffer solution system is composed of glycine buffer solution, MES buffer solution and MOPS buffer solution according to a scientific and reasonable proportion, not only can effectively play a role in buffering the system, but also can better protect components in a product from being influenced by an external environment, and improve the stability and the accuracy of the product.

3. The minimum detection limit of the adiponectin kit is 0.33mg/L, the sensitivity can reach 5mg/L, the linear range is 2-40mg/L, and the adiponectin kit has the characteristics of flexibility and universality.

4. The stabilizer provided by the invention is prepared from bovine serum albumin, casein and mannitol in a ratio, and can effectively improve the stability of a kit system. The stability is used as an important index for keeping the product safe and effective as an in vitro diagnostic reagent, and has important significance on the processes of production, transportation, storage, use and the like of the product.

5. The reaction enhancer consisting of polyethylene glycol 6000 and polyethylene glycol 8000 is added into the R1 reagent, so that the sensitivity of product detection can be effectively improved.

6. The method couples the adiponectin antibody with the latex microsphere through a chemical crosslinking method, has high binding rate, low raw material loss and high stability, and can effectively improve the detection sensitivity.

Drawings

FIG. 1 is a graph showing the stability tendency of adiponectin kits according to example 1 of the present invention and comparative example 1.

FIG. 2 is a LOQ chart of adiponectin kits of example 1 of the present invention and comparative example 1.

Detailed Description

The present invention is further described below with reference to examples, and it will be understood by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the present invention.

Example 1

the R1 reagent is: 30mM of glycine buffer solution, 10mM of MES buffer solution, 10mM of MOPS buffer solution, 2g/L of bovine serum albumin, 2g/L of casein, 1g/L of mannitol, 1g/L of sodium azide, 1.5g/L of Proclin 3001.5 g/L, 60002 g/L of polyethylene glycol, 80002 g/L of polyethylene glycol, and Tween 201.5 g/L, Triton-X1001 g/L;

the R2 reagent is: 35mM of glycine buffer solution, 10mM of MES buffer solution, 15mM of MOPS buffer solution, 1g/L of bovine serum albumin, 1g/L of casein, 3g/L of mannitol, 1g/L of sodium azide, 1.5g/L of Proclin 3001.5 and 1.5mg/L of latex microspheres coupled with human adiponectin monoclonal antibodies; the amount of antibody added was 0.3 mg/L.

The preparation method of the adiponectin kit comprises the following steps:

1) preparation of R1 reagent: mixing all the components in the same container according to the component content of the adiponectin R1 reagent, and uniformly mixing to obtain the adiponectin R1 reagent;

2) preparation of R2 reagent:

activating: taking 40% of the total amount of the buffer solution in the R2 reagent, adding the latex microspheres, uniformly mixing the latex microspheres by stirring in a shaking table at 37 ℃ for 10min, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide according to 0.15g/L, and continuously stirring for 35min to uniformly mix to respectively obtain activated latex microspheres;

marking: adding the human adiponectin antibody into the activated latex microspheres while stirring, and uniformly mixing the mixture for 120min at 37 ℃ by a shaking table to obtain latex microspheres coupled with the human adiponectin antibody;

sealing: adding bovine serum albumin, casein, mannitol, sodium azide and Proclin300 into the latex microspheres coupled with the human adiponectin antibodies, fully dissolving and mixing for 50min, and sealing to obtain sealed latex microspheres;

adding the rest buffer solution into the closed latex microspheres, and uniformly mixing to obtain an R2 reagent;

Example 2

the R1 reagent is: 10mM of glycine buffer solution, 15mM of MES buffer solution, 20mM of MOPS buffer solution, 1g/L of bovine serum albumin, 3g/L of casein, 3g/L of mannitol, 1.5g/L of sodium azide, 1.2g/L of Proclin 3001.2 g/L, 60001 g/L of polyethylene glycol, 80003 g/L of polyethylene glycol, and 201.0 g/L, Triton-X1001.5 g/L of Tween;

the R2 reagent is: 50mM of glycine buffer solution, 5mM of MES buffer solution, 10mM of MOPS buffer solution, 1g/L of bovine serum albumin, 3g/L of casein, 3g/L of mannitol, 1.5g/L of sodium azide, 1.2g/L of Proclin300, and 1.2mg/L of latex microspheres coupled with human adiponectin monoclonal antibodies; the amount of antibody added was 0.4 mg/L.

The preparation method of the adiponectin kit comprises the following steps:

2) preparation of R2 reagent:

activating: taking 50% of the total amount of the buffer solution in the R2 reagent, adding the latex microspheres, uniformly mixing the latex microspheres in a shaking table at 37 ℃ for 15min, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine according to 0.2g/L, continuously stirring the mixture for 30min, and uniformly mixing the mixture to obtain activated latex microspheres respectively;

marking: adding the human adiponectin antibody into the activated latex microspheres while stirring, and uniformly mixing the mixture for 90min in a shaking table at 37 ℃ to obtain latex microspheres coupled with the human adiponectin antibody;

sealing: adding bovine serum albumin, casein, mannitol, sodium azide and Proclin300 into the latex microspheres coupled with the human adiponectin antibodies, fully dissolving and mixing for 40min, and sealing to obtain sealed latex microspheres;

3) and (4) independently subpackaging the R1 reagent and the R2 reagent, and sealing and storing to obtain the adiponectin kit.

Example 3

An adiponectin kit comprises an R1 reagent and an R2 reagent, wherein the solvent is purified water; wherein, the first and the second end of the pipe are connected with each other,

the R1 reagent is: glycine buffer 30mM, MES buffer 25mM, MOPS buffer 15mM, bovine serum albumin 3g/L, casein 1g/L, mannitol 2g/L, sodium azide 1.6g/L, Proclin 3002 g/L, polyethylene glycol 60004 g/L, polyethylene glycol 80001 g/L, Tween 202.5g/L, Triton-X1001.5g/L;

the R2 reagent is: 50mM of glycine buffer solution, 10mM of MES buffer solution, 15mM of MOPS buffer solution, 3g/L of bovine serum albumin, 1g/L of casein, 4g/L of mannitol, 1.3g/L of sodium azide, 1.2g/L of Proclin300, and 1.0mg/L of latex microspheres coupled with human adiponectin monoclonal antibodies; the amount of antibody added was 0.5 mg/L.

The preparation method of the adiponectin kit comprises the following steps:

2) preparation of R2 reagent:

activating: taking 60% of the total amount of the buffer solution in the R2 reagent, adding the latex microspheres, uniformly mixing the latex microspheres by stirring in a shaking table at 37 ℃ for 12min, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide according to 0.1g/L, and continuously stirring for 35min to uniformly mix to respectively obtain activated latex microspheres;

marking: adding the human adiponectin antibody into the activated latex microspheres while stirring, and uniformly mixing the mixture for 60min at 37 ℃ by using a shaking table to obtain latex microspheres coupled with the human adiponectin antibody;

sealing: adding bovine serum albumin, casein, mannitol, sodium azide and Proclin300 into the latex microspheres coupled with the adiponectin antibodies, fully dissolving and mixing for 50min, and sealing to obtain sealed latex microspheres;

Example 4

the R1 reagent is: glycine buffer 50mM, MOPS buffer 20mM, bovine serum albumin 1.5g/L, casein 2g/L, mannitol 1.5g/L, sodium azide 1.2g/L, Proclin 3001.5 g/L, polyethylene glycol 60001 g/L, polyethylene glycol 80003 g/L, Tween 201.5 g/L, Triton-X1001.5 g/L;

the R2 reagent is: 50mM of glycine buffer solution, 25mM of MOPS buffer solution, 1g/L of bovine serum albumin, 3g/L of casein, 2g/L of mannitol, 2g/L of sodium azide, 1.5g/L of Proclin 300and 1.6mg/L of latex microspheres coupled with human adiponectin monoclonal antibodies; the amount of antibody added was 0.3 mg/L.

The preparation method of the adiponectin kit comprises the following steps:

2) preparation of R2 reagent:

activating: taking 40% of the total amount of the buffer solution in the R2 reagent, adding the latex microspheres, uniformly mixing the latex microspheres by stirring in a shaking table at 37 ℃ for 10min, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide according to 0.12g/L, and continuously stirring for 35min to uniformly mix to respectively obtain activated latex microspheres;

marking: adding the human adiponectin antibody into the activated latex microspheres while stirring, and uniformly mixing the mixture for 120min in a shaking table at 37 ℃ to obtain latex microspheres coupled with the human adiponectin antibody;

Example 5

the R1 reagent is: 40mM of glycine buffer solution, 25mM of MES buffer solution, 2.5g/L of bovine serum albumin, 2.5g/L of casein, 3g/L of mannitol, 1.5g/L, Proclin 3001 g/L of sodium azide, 60001 g/L of polyethylene glycol, 80003 g/L, Tween 202, 202 g/L, Triton-X1002.5g/L of polyethylene glycol;

the R2 reagent is: 45mM of glycine buffer solution, 20mM of MES buffer solution, 1g/L of bovine serum albumin, 3g/L of casein, 3g/L of mannitol, 1.3g/L of sodium azide, 1.1g/L of Proclin 300and 2mg/L of latex microspheres coupled with human adiponectin monoclonal antibodies; the amount of antibody added was 0.4 mg/L.

The preparation method of the adiponectin kit comprises the following steps:

2) preparation of R2 reagent:

activating: taking 40% of the total amount of the buffer solution in the R2 reagent, adding the latex microspheres, uniformly mixing the latex microspheres by stirring in a shaking table at 37 ℃ for 10min, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide according to 0.18g/L, and continuously stirring for 35min to uniformly mix to respectively obtain activated latex microspheres;

sealing: adding bovine serum albumin, casein, mannitol, sodium azide and Proclin300 into the latex microsphere coupled with the human adiponectin antibody, fully dissolving and mixing for 50min, and sealing to obtain a sealed latex microsphere;

Comparative example 1

the R1 reagent is: 80mM of glycine buffer solution, 2g/L, Proclin 3005 g/L of bovine serum albumin, 60008g/L, Tween 206 g/L of polyethylene glycol;

the R2 reagent is: 80mM of glycine buffer solution, 2g/L, Proclin 3005/3005 g/L of bovine serum albumin and 2mg/L of latex microspheres coupled with the human adiponectin monoclonal antibody; the amount of antibody added was 0.1 mg/L.

The preparation method of the adiponectin kit comprises the following steps:

2) preparation of R2 reagent:

activating: taking 40% of the total amount of the glycine buffer solution, adding the latex microspheres, uniformly mixing the mixture in a shaker at 37 ℃ for 10min, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine according to 0.15g/L, and continuously stirring the mixture for 35min to uniformly mix the mixture to respectively obtain activated latex microspheres;

sealing: adding bovine serum albumin and Proclin300 into the latex microspheres coupled with the human adiponectin antibodies, fully dissolving and mixing for 50min, and sealing to obtain sealed latex microspheres;

adding the residual glycine buffer solution into the closed latex microspheres, and uniformly mixing to obtain an R2 reagent;

The adiponectin kits prepared in the examples and comparative examples of the invention were subjected to the following performance tests:

1. sensitivity (minimum detection limit) test

The reagents prepared in examples 1 to 5 and comparative example 1 were used, and the test was performed using a zero-concentration reference substance as a sample, the measurement was repeated 10 times, the average value X and the standard deviation SD of the results of the 10 measurement concentration values were calculated, and X +2SD was used as the minimum detection limit of the reagent. The test results are shown in Table 1, and the lowest detection limit of the reagent is 0.33mg/L and lower than 1.5mg/L, which meets the requirements.

TABLE 1 results of sensitivity test of the kit prepared in example 1

Number of detections	1	2	3	4	5	6	7	8	9	10	X	SD	X+2SD
														Example 1	0.18	0.27	0.19	0.00	0.26	0.10	0.00	0.15	0.00	0.08	0.12	0.10	0.33
Example 2	0.18	0.29	0.26	0.03	0.28	0.15	0.00	0.22	0.02	0.10	0.15	0.11	0.38
														Example 3	0.19	0.37	0.32	0.08	0.37	0.17	0.07	0.32	0.05	0.18	0.21	0.12	0.46
Example 4	0.24	0.37	0.32	0.16	0.37	0.18	0.10	0.41	0.15	0.20	0.25	0.10	0.47
														Example 5	0.30	0.47	0.36	0.25	0.47	0.21	0.19	0.43	0.19	0.27	0.31	0.10	0.53
Comparative example 1	1.58	0.48	1.84	1.39	1.46	0.76	0.57	1.87	1.20	1.13	1.23	0.49	2.22

As can be seen from Table 1, the lowest detection limit of the adiponectin kits of examples 1 to 5 of the present invention can reach 0.33 mg/L.

2. Linear range test

Test samples were obtained by dilution of high concentration (40mg/L) samples with low concentration (2mg/L) samples in a multiple ratio, formulated as in Table 2 below. The concentration and volume unit of each sample are unified, and a liquid transfer device with good precision and accuracy is selected when liquid is sucked; the sample is mixed thoroughly during preparation and evaporation or other conditions that degrade the sample are avoided. The results are shown in Table 3.

TABLE 2 sample dilution by fold

Sample number	1	2	3	4	5	6
							Low concentration serum (mL)	1.00	0.80	0.60	0.40	0.20	0.00
High concentration serum (mL)	0.00	0.20	0.40	0.60	0.80	1.00

The linear range assay was performed on the kits prepared in example 1 and comparative example 1, and the concentration of each diluted sample was measured 3 times per sample using a biochemical analyzer.

TABLE 3 Linear Range measurements

As can be seen from Table 3, the linear range result of the reagent in the embodiment 1 of the invention meets the requirement of reagent detection, and the linear correlation coefficient (r) is more than or equal to 0.990 within [2, 40] mg/L; the absolute deviation of linearity is not more than plus or minus 0.5mg/L, and the relative deviation of linearity is not more than plus or minus 2%. The reactivity of the reagent of comparative example 1 was too low, the deviation was large, and the linear range was narrow.

3. Stability test

The adiponectin kits of the embodiment 1 and the comparative example 1 are respectively adopted to carry out detection in 0 th month, 3 rd month, 6 th month, 9 th month, 12 th month, 15 th month and 18 th month under the storage condition of 2-8 ℃, 5 times of detection are carried out on each sample, and an average value is taken. The stability test results are shown in tables 4 and 5, and fig. 1 is a stability trend chart of example 1 and comparative example 1.

Table 4 test results of stability of the kit of example 1

TABLE 5 test results on stability of the kit of comparative example 1

As shown by the stability results of the example 1 and the comparative example 1 in tables 4 and 5 and FIG. 1, the trend of the reagent of the example 1 stored for 18 months at 2-8 ℃ is not significant, namely the reagent of the example 1 stored for 18 months at 2-8 ℃, the change trend of the detection result is not significant and the detection result is in a stable state. And the reagents p of the comparative examples 1-2 are all less than 0.05, the change trend of the detection result is obvious and is in an unstable state, which shows that the addition of the stabilizer is important to the stability of the reagents.

4. Precision test

Adiponectin serum samples at concentrations of 4.25mg/L and 9.25mg/L were tested 10 times using the adiponectin test kits of examples 1 to 5 and comparative example 1, respectively, and the mean (M), Standard Deviation (SD), and Coefficient of Variation (CV) were calculated, and the test results are shown in tables 6 and 7 below.

TABLE 6 precision test results (4.25mg/L)

TABLE 7 precision test results (9.25mg/L)

From tables 6 and 7, the precision of the two levels of the samples of examples 1 to 5 of the invention is high, the repeatability is good, the requirement of reagent detection is met, the coefficient of variation CV is about 2.4 to 2.8 percent, while the kit of comparative example 1 has poor precision, the coefficient of variation CV is high and is 4 percent.

5. LOQ verification test for quantitative detection limit

The quantitative detection limit LOQ is the minimum amount of analyte in a sample which can be quantitatively detected under the defined acceptable conditions, the LOQ of the reagent of example 1 and the LOQ of the reagent of comparative example 1 are verified and compared, the detection results are shown in the following tables 8 and 9, and the LOQ chart of the adiponectin kit is shown in FIG. 2.

Table 8 LOQ validation data for example 1

TABLE 9 LOQ validation data for comparative example 1

As is clear from tables 8 and 9 and FIG. 2, the LOQ of example 1 of the present invention reached 0.7mg/L, while that of comparative example was 1.1mg/L, and example 1 had a lower LOQ than comparative example 1, indicating that the present invention has a higher sensitivity.

Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. An adiponectin kit, characterized in that: comprises an R1 reagent and an R2 reagent, and the solvent is purified water; wherein, the first and the second end of the pipe are connected with each other,

the R1 reagent comprises: 25-100 mM of buffer solution A, 3-11 g/L of stabilizer A, 2-4 g/L of preservative A, 2-7 g/L of reaction enhancer and 2-5 g/L of surfactant;

2. The adiponectin kit according to claim 1, wherein: the buffer solution A and the buffer solution B are combined by two or three of glycine buffer solution, MES buffer solution and MOPS buffer solution.

3. The adiponectin kit according to claim 2, wherein: the buffer solution A and the buffer solution B comprise the following components: 10-50 mM glycine buffer solution, 5-25 mM MES buffer solution and 10-25 mM MOPS buffer solution.

4. The adiponectin kit according to claim 1, wherein: the stabilizer A and the stabilizer B comprise the following components: 1-3 g/L bovine serum albumin, 1-3 g/L casein and 1-5 g/L mannitol.

5. The adiponectin kit according to claim 1, wherein: the preservative A and the preservative B comprise the following components: 1-2 g/L, Proclin 3001-2 g/L sodium azide.

6. The adiponectin kit according to claim 1, wherein: the reaction enhancer in the R1 reagent comprises the following components: polyethylene glycol 60001-4 g/L and polyethylene glycol 80001-3 g/L.

7. The adiponectin kit according to claim 1, wherein: the surfactant components in the R1 reagent are as follows: tween 201-2.5 g/L, Triton-X1001-2.5 g/L.

8. The adiponectin kit according to claim 1, wherein: the adiponectin antibody is a human adiponectin monoclonal antibody, and the addition amount of the antibody is 0.1-0.5 mg/L.

9. The adiponectin kit according to any one of claims 1 to 8, wherein: the preparation method comprises the following steps:

2) preparation of R2 reagent:

marking: adding the adiponectin antibodies into the activated latex microspheres while stirring, and uniformly mixing the activated latex microspheres and the adiponectin antibodies in a shaking table at 37 ℃ for 60-120min to obtain latex microspheres coupled with the adiponectin antibodies;

3) and (3) separately subpackaging the reagent R1 and the reagent R2, and sealing and storing to obtain the adiponectin kit.

10. The adiponectin kit according to claim 9, wherein: the coupling agent is 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine, and the addition amount of the coupling agent is 0.1-0.2 g/L.