CN115097141B

CN115097141B - Adiponectin kit and preparation method thereof

Info

Publication number: CN115097141B
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: 2023-10-20
Anticipated expiration: 2042-06-21
Also published as: CN115097141A

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, and 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 microsphere coupled with adiponectin antibody. The adiponectin kit has the advantages of high sensitivity, good stability, high accuracy, wide linear range, and more than 18 months of cold storage and preservation validity period, is simple and convenient to operate, can be used for clinical detection, and can be used for preventing and treating diseases such as obesity, diabetes, coronary heart disease, cardiovascular diseases and the like.

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. Human studies have found that adiponectin levels can be predictive of the development of type II diabetes and coronary heart disease and show potential against diabetes, atherosclerosis and inflammation in clinical trials.

Human adiponectin comprises 244 amino acids, including 3 regions: amino terminal signal sequence, collagen domain and carboxy terminal globular domain. Adiponectin exists in blood as three subtypes, trimer, hexamer and high molecular weight polymer. Clinical studies show that adiponectin is closely related to type II diabetes, coronary heart disease and insulin resistance, and has the characteristics of resisting atherosclerosis, inflammation and intimal hyperplasia after vascular injury. According to the international diabetes union, there are currently more than 3 hundred million diabetics worldwide, and 46% of patients are not found until after serious diabetic complications occur. The adiponectin kit can detect the adiponectin level in human blood, can provide valuable information for diseases such as diabetes, and can realize early discovery and early treatment of diabetes, thereby reducing the incidence of diabetes.

Currently, the detection methods of adiponectin mainly include a chemiluminescent method, an enzyme-linked immunoassay method, a radioimmunoassay method and a latex immunoturbidimetry method. Chemiluminescent methods, while sensitive and simple, have poor selectivity, and react to a range of compounds, rather than to a single compound, with poor specificity. The ELISA method has higher sensitivity, but has complicated 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 by manual operation and external factors, low degree of automation and the like, and the radioactive marker can harm operators and pollute the environment. The latex-enhanced immunoturbidimetry has the advantages of rapidness, simplicity, convenience, high precision, easiness in automation, suitability for simultaneous detection of large-scale specimens and the like, and is widely applied by people. 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 is insufficient or the production efficiency is low and the cost is high.

In order to promote popularization and application of an 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 above purpose, the present invention provides the following scheme:

an adiponectin kit comprises an R1 reagent and an R2 reagent, wherein a solvent is purified water; 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 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 microsphere coupled with adiponectin antibody.

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

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

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

Preferably, the components of the preservative A and the preservative B are as follows: 1-2 g/L, proclin of sodium azide and 300-2 g/L.

Preferably, the reaction enhancer component in the R1 reagent is: polyethylene glycol 6000-4 g/L and polyethylene glycol 80001-3 g/L.

Preferably, the surfactant component in the R1 reagent is: tween 20-2.5 g/L, triton-X100-2.5 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 240nm.

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:

(1) activating: adding 40-60% of the total amount of the buffer solution B into latex microspheres, stirring at 37 ℃ for 10-15min, adding a coupling agent, continuously stirring for 30-35min, and uniformly mixing to obtain activated latex microspheres respectively;

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

(3) closing: adding a preservative B and a stabilizer B into the latex microsphere coupled with the adiponectin antibody, fully dissolving and mixing for 40-60min, and sealing to obtain a sealed latex microsphere;

(4) adding the rest 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 preserving to obtain the adiponectin kit.

Specifically, the coupling agent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide, and the addition amount of the coupling agent is 0.1-0.2g/L. The coupling agent is added to activate the latex microsphere, so that the efficiency of the European-Union reaction is improved.

The adiponectin monoclonal antibody used in the invention is purchased from Nanjing Litui biotechnology Co., ltd, and the latex microsphere is purchased from Japanese JSR Co., with the particle size of 240nm.

The invention has the following beneficial effects:

1. the adiponectin kit has the advantages of high sensitivity, good stability, high accuracy, wide linear range, more than 18 months of storage life at the temperature of 2-8 ℃, simple and convenient operation, can be used for clinical detection, and has positive significance for preventing and treating diseases such as obesity, diabetes, coronary heart disease, cardiovascular diseases and the like.

2. The buffer solution system is composed of glycine buffer solution, MES buffer solution and MOPS buffer solution according to scientific and reasonable proportion, so that the buffer capability of the system can be effectively achieved, components in the product can be well protected from the influence of external environment, and the stability and accuracy of the product are improved.

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

4. The stabilizing agent provided by the invention is prepared from bovine serum albumin, casein and mannitol in proportion, and can effectively improve the stability of a kit system. The stability is used as an important index for keeping the safety and effectiveness of the product in vitro diagnostic reagent, and has important significance for the production, transportation, preservation, use and other processes 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. According to the invention, the adiponectin antibody and the latex microsphere are coupled by a chemical crosslinking method, so that the coupling rate is high, the raw material loss is low, the stability is high, and the detection sensitivity can be effectively improved.

Drawings

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

FIG. 2 is a LOQ diagram of adiponectin kit according to example 1 and comparative example 1 of the present invention.

Detailed Description

The present invention is further described below with reference to examples, which are only for illustration of the present invention and should not be construed as limiting the scope of the present invention as will be understood by those skilled in the art.

Example 1

the R1 reagent is as follows: 30mM glycine buffer, 10mM MES buffer, 10mM MOPS buffer, 2g/L bovine serum albumin, 2g/L casein, 1g/L mannitol, 1g/L, proclin sodium azide, 1.5g/L polyethylene glycol 6000, 8000 2g/L, tween polyethylene glycol 20, 1.5g/L, triton-X100 g/L;

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

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

2) Preparation of R2 reagent:

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

(2) marking: adding the human adiponectin antibody into the activated latex microsphere while stirring, and stirring for 120min at 37 ℃ by a shaking table to obtain the latex microsphere coupled with the human adiponectin antibody;

(3) closing: 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 for sealing to obtain a sealed latex microsphere;

(4) adding the rest buffer solution into the closed latex microsphere, and uniformly mixing to obtain an R2 reagent;

Example 2

the R1 reagent is as follows: glycine buffer 10mM, MES buffer 15mM, MOPS buffer 20mM, bovine serum albumin 1g/L, casein 3g/L, mannitol 3g/L, sodium azide 1.5g/L, proclin 300.2 g/L, polyethylene glycol 6000 1g/L, polyethylene glycol 8000 3g/L, tween 20.0 g/L, triton-X100.5 g/L;

the R2 reagent is as follows: glycine buffer solution 50mM, MES buffer solution 5mM, MOPS buffer solution 10mM, bovine serum albumin 1g/L, casein 3g/L, mannitol 3g/L, sodium azide 1.5g/L, proclin 300.2 g/L, latex microsphere coupled with human adiponectin monoclonal antibody 1.2mg/L; the amount of antibody added was 0.4mg/L.

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

2) Preparation of R2 reagent:

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

(2) marking: adding the human adiponectin antibody into the activated latex microsphere while stirring, and stirring for 90min at 37 ℃ by a shaking table to obtain the latex microsphere coupled with the human adiponectin antibody;

(3) closing: 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 40min for sealing to obtain a sealed latex microsphere;

Example 3

the R1 reagent is as follows: 30mM glycine buffer, 25mM MES buffer, 15mM MOPS buffer, 3g/L bovine serum albumin, 1g/L casein, 2g/L mannitol, 1.6g/L, proclin sodium azide, 300 g/L polyethylene glycol 6000, 80001 g/L, tween 20.5 g/L, triton-X100.5 g/L polyethylene glycol;

the R2 reagent is as follows: glycine buffer solution 50mM, MES buffer solution 10mM, MOPS buffer solution 15mM, bovine serum albumin 3g/L, casein 1g/L, mannitol 4g/L, sodium azide 1.3g/L, proclin 300.2 g/L, latex microsphere coupled with human adiponectin monoclonal antibody 1.0mg/L; the amount of antibody added was 0.5mg/L.

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

2) Preparation of R2 reagent:

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

(2) marking: adding the human adiponectin antibody into the activated latex microsphere while stirring, and stirring for 60min at 37 ℃ by a shaking table to obtain the latex microsphere coupled with the human adiponectin antibody;

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

Example 4

the R1 reagent is as follows: 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 300.5 g/L, polyethylene glycol 6000 1g/L, polyethylene glycol 8000 3g/L, tween 20.5 g/L, triton-X100.5 g/L;

the R2 reagent is as follows: glycine buffer solution 50mM, MOPS buffer solution 25mM, bovine serum albumin 1g/L, casein 3g/L, mannitol 2g/L, sodium azide 2g/L, proclin 300.5 g/L, latex microsphere coupled with human adiponectin monoclonal antibody 1.6mg/L; the amount of antibody added was 0.3mg/L.

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

2) Preparation of R2 reagent:

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

Example 5

the R1 reagent is as follows: glycine buffer 40mM, MES buffer 25mM, bovine serum albumin 2.5g/L, casein 2.5g/L, mannitol 3g/L, sodium azide 1.5g/L, proclin300 g/L, polyethylene glycol 6000 1g/L, polyethylene glycol 8000 3g/L, tween20 g/L, triton-X100.5 g/L;

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

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

2) Preparation of R2 reagent:

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

Comparative example 1

the R1 reagent is as follows: glycine buffer 80mM, bovine serum albumin 2g/L, proclin, 5g/L, polyethylene glycol 60008g/L, tween, 20 g/L;

the R2 reagent is as follows: 80mM glycine buffer, 2g/L, proclin of bovine serum albumin, 5g/L and 2mg/L of latex microsphere coupled with human adiponectin monoclonal antibody; the amount of antibody added was 0.1mg/L.

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

2) Preparation of R2 reagent:

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

(3) closing: adding bovine serum albumin and Proclin300 into the latex microsphere coupled with the human adiponectin antibody, fully dissolving and mixing for 50min for sealing to obtain a sealed latex microsphere;

(4) adding the rest glycine buffer solution into the closed latex microsphere, and uniformly mixing to obtain an R2 reagent;

The adiponectin kit prepared in the examples and comparative examples of the present invention was subjected to the following performance tests:

1. sensitivity (minimum detection limit) test

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

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

Number of times of detection	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 minimum limit of detection of adiponectin in the adiponectin kits according to examples 1 to 5 of the invention was 0.33mg/L.

2. Linear range test

Test samples were obtained by subjecting high concentration (40 mg/L) samples to double dilution with low concentration (2 mg/L) samples, and the formulation scheme is shown in Table 2 below. The concentration and volume units of each sample are unified, and a pipetting device with good precision and accuracy is selected when liquid is sucked; the samples are thoroughly mixed during preparation and evaporation or other conditions that deteriorate the samples are avoided. The test results are shown in Table 3.

TABLE 2 sample multiple dilution

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 test kits prepared in example 1 and comparative example 1 were subjected to linear range detection, and the concentration of each diluted sample was measured 3 times with a biochemical analyzer.

TABLE 3 Linear Range detection results

As can be seen from Table 3, the linear range results of the reagent of example 1 of the present invention meet the requirements of reagent detection, and the linear correlation coefficient (r) is not less than 0.990 in [2, 40] mg/L; the absolute deviation of linearity is not more than +/-0.5 mg/L, and the relative deviation of linearity is not more than +/-2%. The reagent of comparative example 1 has too low reactivity, large deviation and narrow linear range.

3. Stability test

The adiponectin kits of example 1 and comparative example 1 were used under storage conditions of 2 to 8℃at month 0, month 3, month 6, month 9, month 12, month 15, and month 18, respectively, and each sample was tested 5 times, and averaged. The stability test results are shown in tables 4 and 5, and fig. 1 is a graph showing the stability trend of example 1 and comparative example 1.

TABLE 4 stability test results for the kit of example 1

TABLE 5 stability test results for 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 reagent of the example 1 was stored at 2-8deg.C for 18 months, and p > 0.05, the trend was not significant, i.e., the reagent of the example 1 was stored at 2-8deg.C for 18 months, the trend of the change of the detection result was not significant, and the reagent was in a stable state. The reagents p in comparative examples 1-2 are all smaller than 0.05, the variation trend of the detection result is remarkable, and the reagent p is in an unstable state, which indicates that the addition of the stabilizer is important for the stability of the reagent.

4. Precision test

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

TABLE 6 precision measurement results (4.25 mg/L)

TABLE 7 precision measurement results (9.25 mg/L)

As can be seen from tables 6 and 7, the two levels of the kit of examples 1 to 5 of the present invention have higher precision and good repeatability, and meet the requirements of reagent detection, and the coefficient of variation CV is about 2.4 to 2.8%, while the kit of comparative example 1 has poorer repeatability, and the coefficient of variation CV is higher, being 4%.

5. Quantitative detection limit LOQ verification test

The limit of detection of the LOQ is the minimum amount of analyte in the sample detected quantitatively under a predetermined acceptable condition, and the LOQ of the reagent of example 1 and that of comparative example 1 are compared by verification, and the detection results are shown in tables 8 and 9 below, and the LOQ diagram of the adiponectin kit is shown in fig. 2.

Table 8 LOQ verification data of example 1

Table 9 LOQ verification data for comparative example 1

As can be seen from tables 8, 9 and FIG. 2, the LOQ of example 1 of the present invention can reach 0.7mg/L, whereas the LOQ of comparative example is 1.1mg/L, and example 1 has a lower LOQ than comparative example 1, indicating that the present invention has higher sensitivity.

While the foregoing describes the embodiments of the present invention, it should be understood that the present invention is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.

Claims

1. An adiponectin kit characterized in that: the reagent comprises an R1 reagent and an R2 reagent, and the solvent is purified water; wherein,,

the R1 reagent is as follows: 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 reaction enhancer comprises the following components: polyethylene glycol 6000 to 4g/L and polyethylene glycol 8000 to 3 g/L; the surfactant comprises the following components: tween 20-2.5 g/L and Triton-X100-2.5 g/L;

the R2 reagent is as follows: 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 microsphere coupled with adiponectin antibody; the adiponectin antibody is a human adiponectin monoclonal antibody, and the addition amount of the antibody is 0.1-0.5 mg/L;

the buffer solution A and the buffer solution B comprise the following components: glycine buffer solution 10-50 mM, MES buffer solution 5-25 mM and MOPS buffer solution 10-25 mM; the components of the stabilizer A and the stabilizer B are as follows: bovine serum albumin 1-3 g/L, casein 1-3 g/L and mannitol 1-5 g/L.

2. The adiponectin kit according to claim 1, wherein: the components of the preservative A and the preservative B are as follows: 1-2 g/L of sodium azide and 300-2 g/L of Proclin.

3. The adiponectin kit according to claim 1 or 2, characterized in that: the preparation method comprises the following steps:

2) Preparation of R2 reagent:

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

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