Adiponectin determination kit and preparation method thereof

Abstract

The invention provides an adiponectin determination kit and a preparation method thereof, the kit comprises a reagent R1 and a reagent R2, the reagent R1 comprises a buffer solution, a preservative, a stabilizer, an unwinding agent, an anti-human adiponectin antibody and Protein; the reagent R2 comprises a buffer solution, a preservative, a stabilizer and nano microspheres. According to the kit, hexamer and multimer of adiponectin can be dissociated into trimer, the trimer is combined with a specific antibody after being captured by Protein, the antibody is connected with nano microspheres in a physical adsorption mode in the binding reaction process of the antibody and the adiponectin trimer, turbidity is formed, and then the content of total adiponectin in a sample to be detected is accurately determined. In the preparation process of the kit disclosed by the invention, the adiponectin antibody and the nano-microspheres do not need to be chemically coupled to form sensitization particles, and disulfide bonds formed between the antibody and the nano-microspheres are prevented from being dissociated by the unwinding agent, so that the use of a scavenger of the unwinding agent can be avoided, and the kit is simple and convenient to prepare.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnostic reagent preparation, and specifically relates to an adiponectin determination kit and a preparation method thereof. Background Art

[0002] Adiponectin is an endogenous active glycoprotein mainly secreted by adipocytes. It is a key regulatory factor involved in glucose and lipid metabolism. In clinical trials, it was found that adiponectin levels can predict the development of type 2 diabetes and coronary heart disease, and in clinical trials, it has shown anti-diabetic, anti-atherosclerotic and anti-inflammatory potential. Adiponectin can enhance the sensitivity of muscles and liver to insulin, regulate glucose and lipid metabolism, help prevent and treat type 2 diabetes, improve endothelial function, inhibit the proliferation and migration of vascular smooth muscle cells, and reduce the formation of atherosclerosis, thus having a cardiovascular protective effect, and thus becoming an important target for metabolic diseases, cardiovascular diseases and inflammatory diseases.

[0003] Adiponectin exists in human serum in three structural forms, including low molecular weight trimer (LMW, about 90KDa), medium molecular weight hexamer (MMW, about 180KDa) and high molecular weight polymer (HMW about 360-540KDa). The formation of hexamer depends on the disulfide bond mediated by the cysteine ​​residue at position 39 (CYS-39), while the polymer depends on the formation of complete hexamer. Therefore, both polymers are formed by adiponectin trimer monomers through disulfide bonds to obtain complex stability.

[0004] At present, the main methods used in clinical practice are enzyme-linked immunosorbent assay, fluorescent immunoassay and immunoturbidimetry. Enzyme-linked immunosorbent assay has low requirements for instruments and detection environment, but its solid phase material is a microplate with a limited number of micropores, and multiple washing and separation are required during the detection process, the detection time is long, and the precision and accuracy are low. Fluorescent immunoassay uses fluorescent substances as immune markers to bind to antibodies. After the antigen-antibody specific binding, an immune complex is formed. The fluorescence intensity is measured by a fluorescent detector to judge the concentration of the antigen. This method has high sensitivity, but is easily affected by fluorescent substances, has complex operations, and has poor stability. Immunoturbidimetry is based on the basic principle of specific binding between antigens and antibodies for detection. However, since adiponectin exists in the form of polymers of different subtypes in the human body, and antibodies can only specifically recognize a specific site on adiponectin, different subtypes cannot be identified, and the adiponectin concentration of the patient cannot be truly reflected, resulting in deviations in the test results and affecting clinical judgments. Summary of the invention

[0005] The invention discloses an adiponectin determination kit and a preparation method thereof, so as to solve the problems of inconvenient preparation and inaccurate detection results in the prior art.

[0006] The first aspect of the present invention provides an adiponectin assay kit, comprising: reagent R1 and reagent R2, wherein the reagent R1 comprises a buffer, a preservative, a stabilizer, a detethering agent, an anti-human adiponectin antibody and a protein; and the reagent R2 comprises a buffer, a preservative, a stabilizer and nanoparticles.

[0007] Furthermore, the ratio of each component in the reagent R1 is as follows:

[0008]

[0009] Furthermore, the ratio of each component in the reagent R2 is as follows:

[0010]

[0011] Furthermore, the buffer is selected from at least one of 2-(N-morpholine)ethanesulfonic acid (MES), 3-(N-morpholine)-2-hydroxypropanesulfonic acid (MOPSO), 3-(N-morpholine)propanesulfonic acid (MOPS), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), glycine, and phosphate.

[0012] Furthermore, the preservative is selected from at least one of sodium azide, thimerosal, sodium benzoate, parabens, and Proclin300.

[0013] Furthermore, the stabilizer is selected from at least one of bovine serum albumin (BSA), trehalose, mannitol, glucose and glycerol.

[0014] Furthermore, the unwinding agent is selected from at least one of dithiothreitol (DTT), β-mercaptoethanol (β-ME), cysteine ​​(CYS), glutathione (GSH), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and sodium thiosulfate (Na2S2O3), and its function is to destroy the disulfide bonds in the adiponectin hexamer and polymer, so as to unwind it into adiponectin trimer.

[0015] Furthermore, the anti-human adiponectin antibody is a monoclonal antibody or a polyclonal antibody, which functions to specifically react with the adiponectin trimer in the sample to be tested, thereby causing a change in absorbance in the solution.

[0016] Furthermore, the protein is selected from at least one of Protein A derived from Staphylococcus aureus, Protein G derived from Streptococcus, and Protein L derived from Streptococcus Davidii, and its function is to capture the adiponectin trimer so that it can quickly and specifically bind to the anti-human adiponectin antibody in reagent 1.

[0017] Furthermore, the nanospheres are selected from any one of polystyrene microspheres, magnetic nanospheres, colloidal gold microspheres, and composite nanospheres, and their function is to couple with the anti-human adiponectin antibody by physical adsorption during the reaction process, thereby increasing the signal during the reaction process and improving sensitivity and accuracy.

[0018] The second aspect of the present invention provides a method for preparing an adiponectin assay kit, comprising:

[0019] According to the required volume of reagent 1, calculate and weigh according to the following ratio: buffer 10-500mmol / L, preservative 0.05%-0.5%, stabilizer 0.1%-5%, unwinding agent 0.1%-5%, anti-human adiponectin antibody 20-200mg / L, 5-100mg / L, dissolve in the corresponding volume of purified water to obtain reagent R1;

[0020] According to the required volume of reagent 2, calculate and weigh according to the following ratio: buffer solution 10-500mmol / L, preservative 0.05%-0.5%, stabilizer 0.1%-5%, unwinding agent scavenger 0.01%-1%, nano-microspheres 0.01%-1%, dissolve in the corresponding volume of purified water to obtain reagent R2.

[0021] The present invention provides an adiponectin assay kit, which improves detection accuracy and anti-interference ability mainly through the following aspects: first, a reducing substance is added to reagent 1 to destroy the disulfide bonds in adiponectin polymers, so that hexamers and polymers are unwound into adiponectin trimers; second, protein is added to reagent 1, and anti-human adiponectin antibodies react specifically with adiponectin trimers (including original adiponectin trimers and adiponectin trimers formed by unwound) in a test object to prevent them from being multimerized again, and protein connects the anti-human adiponectin antibodies to increase the affinity between the anti-human adiponectin antibodies and nano-microspheres; third, nano-microspheres are added to reagent 2, and during the test process, the volume of immune complexes in the reaction system is increased by a physical adsorption method with the anti-human adiponectin antibodies, thereby amplifying immune signals, and the non-covalent bonds formed between the anti-human adiponectin antibodies and the nano-microspheres are not affected by the unwound agent in reagent 1, and there is no need to add a scavenger to reagent 2 to remove the unwound agent.

[0022] In the preparation method of an adiponectin assay kit provided by the present invention, anti-human adiponectin antibodies do not need to be coupled with nano-microspheres in advance to form sensitized particles. After the adiponectin hexamers and adiponectin polymers to be tested are decomposed into trimers, the original adiponectin trimers are captured by proteins, specifically react with the anti-human adiponectin antibodies in reagent 1, and physically adsorb with the nano-microspheres in reagent 2 to increase the reaction signal.

[0023] The beneficial effects of the present invention are as follows: In most inventions, the trimer formed after the dissociation of the adiponectin hexamer or polymer in reagent 1 is formed, and then reagent 2 is added to allow the antibody to bind to the adiponectin trimer. During this reaction, a quencher of the unwinding agent needs to be added to reagent 2 to prevent excessive dissociation, so that the trimer is further dissociated into monomers, and reacts with the adiponectin trimer through the latex particles of the coupled antibody in reagent 2. The unwinding agent in reagent 1 is not specific, and there is at least a 5-minute interval before adding reagent 2. If the adiponectin trimer formed after the unwinding of the adiponectin hexamer and polymer in reagent 1 is not protected in time, it will also be further dissociated into monomers before the quencher is added. The kit provided by the present invention adds Protein A to reagent 1, and Protein A captures the dissociated adiponectin trimer at any time and then quickly combines with the anti-human adiponectin antibody to avoid further dissociation. After reagent 2 is added, the antibody combines with the nanoparticles by physical adsorption to amplify the immune signal, and the adiponectin content in the sample is calculated by the change in absorbance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The comparison results of the self-prepared reagent Example 1 and the purified adiponectin trimer;

[0025] Figure 2 The comparison results of the self-prepared reagent Example 1 and the purified adiponectin hexamer;

[0026] Figure 3 The comparison results of the self-prepared reagent Example 1 and the purified adiponectin multimer;

[0027] Figure 4 The comparison results of serum adiponectin concentrations in Examples 1-4 are shown;

[0028] Figure 5 The comparison results of serum adiponectin concentrations between Example 1 and Comparative Examples 1-3 are shown;

[0029] Figure 6 This is the quality control deviation diagram of self-prepared reagent Example 1 after opening the bottle for 30 days;

[0030] Figure 7 This is the quality control deviation diagram of self-prepared reagent Example 1 heat-treated at 37°C for 10 days. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] Reagent 1: 50 mmol / L 2-(N-morpholino)ethanesulfonic acid (pH 6.5), 0.1% Proclin 300, 0.5% bovine serum albumin, 1% dithiothreitol, 50 mg / L anti-human adiponectin monoclonal antibody, 20 mg / L Protein A;

[0034] Reagent 2: 50 mmol / L 2-(N-morpholino)ethanesulfonic acid (pH 6.5), 0.1% Proclin 300, 0.5% bovine serum albumin, and 0.05% polystyrene microspheres.

[0035] Example 2

[0036] Reagent 1: 150 mmol / L 3-(N-morpholino)-2-hydroxypropanesulfonic acid (pH 7.5), 0.2% sodium azide, 3% trehalose, 1% cysteine, 1% β-mercaptoethanol, 50 mg / L anti-human adiponectin monoclonal antibody, 20 mg / L Protein G;

[0037] Reagent 2: 150 mmol / L 3-(N-morpholine)-2-hydroxypropanesulfonic acid (pH 7.5), 0.2% sodium azide, 3% trehalose, and 0.5% magnetic nanospheres.

[0038] Example 3

[0039] Reagent 1: 500 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (pH 8.5), 0.4% sodium benzoate, 0.1% p-hydroxybenzoate, 3% glucose, 2% glycerol, 1% tris(2-carboxyethyl)phosphine hydrochloride, 4% sodium thiosulfate, 200 mg / L anti-human adiponectin monoclonal antibody, 100 mg / L Protein L;

[0040] Reagent 2: 500 mmol / L 3-(N-morpholine)-2-hydroxypropanesulfonic acid (pH 7.5), 0.4% sodium benzoate, 0.1% p-hydroxybenzoate, 3% glucose, 2% glycerol, and 1% composite nanospheres.

[0041] Example 4

[0042] Reagent 1: 10 mmol / L 3-(N-morpholino)propanesulfonic acid (pH 6.5), 0.05% thimerosal, 0.5% mannitol, 0.1% glutathione, 20 mg / L anti-human adiponectin polyclonal antibody, 5 mg / L Protein A;

[0043] Reagent 2: 10 mmol / L 3-(N-morpholino)propanesulfonic acid (pH 6.5), 0.05% thimerosal, 0.5% mannitol, and 0.01% colloidal gold microspheres.

[0044] Comparative Example 1

[0045] Compared with Example 1, the only difference of Comparative Example 1 is that the reagent R1 does not contain Protein A and dithiothreitol.

[0046] Comparative Example 2

[0047] Compared with Example 1, the only difference of Comparative Example 2 is that the reagent R1 does not contain Protein A.

[0048] Comparative Example 3

[0049] Compared with Example 1, the only difference of Comparative Example 3 is that the reagent R1 does not contain dithiothreitol.

[0050] Application of prepared reagents in biochemical analyzers

[0051] Test kit: kits of examples and comparative examples

[0052] Test instrument: Hitachi 7100 biochemical analyzer

[0053] The specific operation is to set the parameters: test method: main wavelength: 570nm; sub-wavelength: 800nm; photometric points: 18-34; sample volume / R1 / R2: 8μL / 160μL / 40μL; calibration method: multi-point calibration.

[0054] Purified adiponectin trimer, adiponectin hexamer and adiponectin polymer of different concentrations were taken and compared with the reagents in Example 1. The results are as follows: Figures 1 to 3 shown.

[0055] From the results, it can be seen that the embodiments of the present invention have a good correlation with adiponectin trimer, adiponectin hexamer and adiponectin polymer, which indicates that the embodiments of the present invention have a high detection accuracy for adiponectin trimer, adiponectin hexamer and adiponectin polymer, and the test results will be closer to the concentration of total adiponectin.

[0056] Example 1-4 Serum adiponectin concentration comparison test results are as follows Figure 4 From the results, it can be seen that within the concentration range of the present invention, the measured values ​​of different embodiments are consistent, and all can accurately measure the concentration of adiponectin in serum.

[0057] The serum adiponectin concentrations of Example 1 and Comparative Examples 1-3 were compared and tested. Figure 5As shown. From the results, it can be seen that the use of protein or unwinding agent alone cannot accurately measure the concentration of adiponectin in serum samples. The two need to cooperate with each other to play a synergistic role and accurately and effectively detect the concentration of adiponectin in serum. The interaction mechanism between protein and unwinding agent is as follows: the unwinding agent first unwinds the adiponectin hexamer and adiponectin polymer, and the unwinding adiponectin trimer and the original adiponectin trimer in the sample to be tested are quickly captured by protein and transferred to the adiponectin antibody, so that the adiponectin trimer reacts specifically with the adiponectin antibody to form an immune complex.

[0058] Opening stability test: Test Example 1 self-prepared reagent opening and self-prepared reagent refrigeration stability, that is, test the consistency between the quality control results and the target value within 30 days after opening the bottle, test once every 5 days, and the stability is used as a reference for relative deviation fluctuations. The test results are as follows Figure 6 shown.

[0059] From the results, it can be seen that within 30 days after the bottle is opened, the relative deviation of the quality control product of the embodiment of the present invention is within ±5%, indicating that the performance of the reagent is stable within 30 days after the bottle is opened, and meets the test requirements.

[0060] Heat treatment stability test: Test Example 1 self-prepared reagent 37 ° C heat treatment for 10 days and self-prepared reagent refrigeration stability, that is, 37 ° C heat treatment within 10 days to test the quality control product results and the target value of the degree of consistency, once every 2 days, the stability is used as a reference relative deviation fluctuation, the test results are as follows Figure 7 shown.

[0061] From the results, it can be seen that the relative deviation of the quality control product of the embodiment of the present invention is within ±5% within 10 days of heat treatment at 37°C, indicating that the performance of the reagent is stable within 10 days of heat treatment at 37°C and meets the test requirements.

[0062] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the scope of the claims of the present invention to be approved.

Claims

1. A kit for determining adiponectin, characterized in that: include: Reagent R1 and reagent R2, wherein the reagent R1 comprises a buffer, a preservative, a stabilizer, a detethering agent, an anti-human adiponectin antibody and a protein; and the reagent R2 comprises a buffer, a preservative, a stabilizer and nano-microspheres.

2. The adiponectin assay kit according to claim 1, characterized in that: The ratio of each component in the reagent R1 is as follows:

3. The adiponectin assay kit according to claim 1, characterized in that: The ratio of each component in the reagent R2 is as follows:

4. The adiponectin assay kit according to claim 1, characterized in that: The buffer is selected from at least one of 2-(N-morpholine)ethanesulfonic acid (MES), 3-(N-morpholine)-2-hydroxypropanesulfonic acid (MOPSO), 3-(N-morpholine)propanesulfonic acid (MOPS), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), glycine, and phosphate.

5. The adiponectin assay kit according to claim 1, characterized in that: The preservative is selected from at least one of sodium azide, thimerosal, sodium benzoate, parabens, and Proclin 300.

6. The adiponectin assay kit according to claim 1, characterized in that: The stabilizer is selected from at least one of bovine serum albumin (BSA), trehalose, mannitol, glucose and glycerol.

7. The adiponectin assay kit according to claim 1, characterized in that: The unwinding agent is selected from at least one of dithiothreitol (DTT), β-mercaptoethanol (β-ME), cysteine ​​(CYS), glutathione (GSH), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and sodium thiosulfate (Na2S2O3).

8. The adiponectin assay kit according to claim 1, characterized in that: The anti-human adiponectin antibody is a monoclonal antibody or a polyclonal antibody.

9. The adiponectin assay kit according to claim 1, characterized in that: The protein is selected from at least one of Protein A derived from Staphylococcus aureus, Protein G derived from Streptococcus, and Protein L derived from Streptococcus Davidii.

10. The adiponectin assay kit according to claim 1, characterized in that: The nano-microspheres are selected from any one of polystyrene microspheres, magnetic nano-microspheres, colloidal gold microspheres, and composite nano-microspheres.

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