Adiponectin antibody magnetic microspheres and adiponectin assay kit

Abstract

The present invention belongs to the technical field of in vitro diagnosis, and discloses an adiponectin antibody magnetic microsphere and an adiponectin assay kit. The adiponectin antibody magnetic microsphere is a streptavidin-coated magnetic microsphere obtained by mixed incubation of functionalized MQ silicone resin-coated magnetic microspheres and streptavidin solution, and mixed incubation with biotin and adiponectin antibody to obtain a biotinylated adiponectin antibody. The adiponectin assay kit includes an adiponectin antibody magnetic microsphere solution, an adiponectin antibody fluorescent marker, and a sample diluent. The adiponectin antibody magnetic microsphere of the present invention is coated in situ with a functional MQ silicone resin to achieve shell protection and surface functionalization modification of magnetic nanoparticles in one step, and is simple to prepare and low in cost. The obtained kit can significantly enhance the effect of immunomagnetic separation and improve detection accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnosis, and specifically relates to an adiponectin antibody magnetic microsphere and an adiponectin determination kit. Background Art

[0002] Immunomagnetic microspheres (IMBs) are a new type of material developed by combining immunology and magnetic carrier technology. IMBs are magnetic microspheres coated with monoclonal antibodies that can specifically bind to target substances containing corresponding antigens to form new complexes. When passing through a magnetic field, this complex can be retained and separated from other components. This process is called immunomagnetic separation. Immunomagnetic separation is simple and easy, has high separation purity, retains the activity of the target substance, and is efficient, rapid, and low in toxicity. It can be widely used in the fields of cell separation and purification, immunoassay, nucleic acid analysis and genetic engineering, and as a carrier for targeted drug release.

[0003] Magnetic microspheres are generally composed of a magnetic core and a protective shell. The main magnetic microspheres currently used are magnetic agarose microspheres, magnetic silica microspheres, magnetic polyacrylamide / polyacrylic acid / polystyrene polymer microspheres, etc. In order to enhance the binding efficiency between magnetic microspheres and coated biopolymers, it is generally necessary to modify the surface of the magnetic microspheres with functional groups. The functional groups on the surface of magnetic microspheres can covalently connect with various active groups of biopolymers such as -OH, -COOH, and -NH2, and can stably fix biologically active substances (such as antibodies, antigens, receptors, enzymes, nucleic acids, and drugs, etc.) on their surface. Therefore, the activity and uniformity of the functional groups on the surface of magnetic microspheres are crucial to the stability of binding and the accuracy of immunoassays.

[0004] CN112877387A discloses that a magnetic material wrapped with SiO2 is used as a magnetic microsphere body, an aminosilane coupling agent is used to chemically modify the magnetic microsphere body, and an amino group is introduced into the outer surface of the magnetic microsphere body to form an amino-modified magnetic microsphere A; an acrylic acid molecule is covalently coupled to the outer surface of the magnetic microsphere A by a covalent reaction between a carboxyl group and an amino group, and a carbon-carbon double bond is introduced to form a magnetic microsphere B containing a carbon-carbon double bond; without adding a crosslinking agent, an acrylic acid monomer molecule is polymerized by a polymerization reaction of a carbon-carbon double bond, and the obtained acrylic acid polymer has a linear main chain and a side chain containing a functional group, and the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain; an acrylic acid polymer-modified magnetic microsphere C is formed; and biotin or a biotin analog is covalently coupled to the end of the polymer side chain through the functional group contained in the polymer side chain to obtain the biomagnetic microsphere. However, the controllability of surface modification using aminosilane coupling agents is poor. They are prone to self-polymerization and cross-linking reactions, which will entrap active groups and reduce the surface modification effect, thereby reducing activity and reactivity, and thus reducing the binding ability with biologically active substances.

[0005] MQ silicone resin is composed of tetrafunctional siloxane chains (SiO 4 / 2 ,Q) and the organosilicon compound containing monofunctional siloxane chain (R3SiO 1 / 2 ,M) through co-hydrolysis-polycondensation reaction of organic silicon compounds. It is a compact spherical object with a double-layer structure, which meets the requirement that magnetic microspheres are uniformly spherical; its spherical core is cage-shaped silica with a high density of silicon-oxygen bonds, which has physiological inertness and strong binding force with inorganic magnetic materials; its spherical shell is R3SiO2 with a low density. 1 / 2 By adjusting the type of capping agent, different active groups can be evenly distributed on the surface. Therefore, it is a very promising work to use MQ silicone resin to simultaneously achieve shell protection and surface functional modification of magnetic microspheres. Summary of the invention

[0006] In view of the shortcomings and deficiencies of the above prior art, the primary purpose of the present invention is to provide a method for preparing adiponectin antibody magnetic microspheres.

[0007] Another object of the present invention is to provide adiponectin antibody magnetic microspheres prepared by the above method.

[0008] Another object of the present invention is to provide an adiponectin assay kit containing the adiponectin antibody magnetic microspheres.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing adiponectin antibody magnetic microspheres comprises the following preparation steps:

[0011] (1) adding silicate, active end-capping agent and magnetic nanoparticles to a solvent and stirring and mixing them uniformly, heating to 60-90° C. under inert atmosphere protection, then dropping an acid catalyst solution to react by stirring, filtering, washing and drying after the reaction is completed to obtain functionalized MQ silicone resin-coated magnetic microspheres;

[0012] (2) mixing and incubating the functionalized MQ silicone resin-coated magnetic microspheres obtained in step (1) with a streptavidin solution to obtain streptavidin-coated magnetic microspheres;

[0013] (3) mixing biotin and adiponectin antibody to react, thereby obtaining biotinylated adiponectin antibody;

[0014] (4) Mixing and incubating the streptavidin-coated magnetic microspheres obtained in step (2) with the biotinylated adiponectin antibody obtained in step (3) to obtain adiponectin antibody magnetic microspheres.

[0015] Furthermore, the silicate in step (1) is at least one of sodium silicate, methyl orthosilicate and ethyl orthosilicate.

[0016] Furthermore, the active end-capping agent in step (1) is at least one of 1,3-bis(aminopropyl)tetramethyldisiloxane, 1,3-bis(3-carboxypropyl)tetramethyldisiloxane, 1,3-bis(3-hydroxypropyl)tetramethyldisiloxane, 1,3-bis(3-glycidylpropyl)tetramethyldisiloxane, 1,3-divinyltetramethyldisiloxane and tetramethyldisiloxane.

[0017] Furthermore, the magnetic nanoparticles in step (1) are selected from magnetic ferrosoferric oxide nanoparticles.

[0018] Furthermore, in step (1), the mass ratio of the silicate, active end-capping agent and magnetic nanoparticles added is 10:0.5-2:10-40.

[0019] Furthermore, the solvent in step (1) is selected to be a solvent that can dissolve or partially dissolve the reaction raw materials silicate and active end-capping agent, but does not dissolve the reaction product MQ silicone resin. Specifically, it can be selected as a mixed solvent of water and small molecule alcohol, such as a mixed solvent of water and ethanol, a mixed solvent of water and isopropanol, etc.; the volume ratio of the water and small molecule alcohol is 4-7:6-3.

[0020] The present invention uses a solvent that can dissolve or partially dissolve the reaction raw materials silicate and active end-capping agent but does not dissolve the reaction product MQ silicone resin for reaction, and adds insoluble magnetic nanoparticles. During the generation of the MQ silicone resin, the core silicon-oxygen bonds thereof have a strong binding force with the inorganic magnetic nanoparticles, and the MQ silicone resin generated by the reaction is precipitated in situ on the surface of the inorganic magnetic nanoparticles, thereby achieving good coating of the magnetic nanoparticles. The product can be separated by simple filtration, and has the advantages of simple process and low cost.

[0021] Furthermore, the acid catalyst solution in step (1) is selected from hydrochloric acid solution or sulfuric acid solution; and the stirring reaction time is 1 to 4 hours.

[0022] Furthermore, the concentration of the streptavidin solution in step (2) is 0.1-1 mg / mL.

[0023] Furthermore, in step (3), the molar ratio of biotin to adiponectin antibody is 1:1-5.

[0024] Furthermore, the adiponectin antibody in step (3) refers to an anti-human adiponectin monoclonal antibody or an anti-human adiponectin polyclonal antibody.

[0025] Furthermore, in step (4), the mass ratio of the streptavidin-coated magnetic microspheres and the biotinylated adiponectin antibody for mixed incubation is 100:5-10.

[0026] Adiponectin antibody magnetic microspheres are prepared by the above method.

[0027] An adiponectin assay kit containing the adiponectin antibody magnetic microspheres comprises an adiponectin antibody magnetic microsphere solution, an adiponectin antibody fluorescent marker and a sample diluent.

[0028] Furthermore, the adiponectin antibody fluorescein marker is an adiponectin antibody acridinium ester marker.

[0029] Furthermore, the sample diluent is a phosphate buffer containing bovine serum albumin, casein, a surfactant and a preservative.

[0030] Furthermore, the adiponectin assay kit also includes a pre-stimulation solution and a stimulation solution; the pre-stimulation solution is a NaOH solution with a concentration of 0.05 to 0.25 mol / L, and the stimulation solution is a H2O2 solution with a concentration of 0.05 to 0.2 mol / L.

[0031] Furthermore, the adiponectin assay kit also includes a calibrator; the calibrator is a phosphate buffer containing adiponectin recombinant antigen, a protein stabilizer and a preservative.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The adiponectin antibody magnetic microspheres of the present invention are coated in situ with functional MQ silicone resin to achieve shell protection and surface functionalization modification of magnetic nanoparticles in one step, which has the advantages of simple preparation process and low cost.

[0034] (2) The adiponectin antibody magnetic microspheres of the present invention have a unique spherical structure of a tight inner layer and a loose outer layer of MQ silicone resin. The inner layer is tightly combined with the inorganic magnetic nanoparticles, and the coating effect is good; the functional groups are evenly distributed in the outermost layer of the microspheres, and the binding efficiency with biologically active substances such as antibodies and antigens is high, which can significantly enhance the effect of immunomagnetic separation and improve the detection accuracy. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0036] Example 1

[0037] A test kit for determining adiponectin in this embodiment includes adiponectin antibody magnetic microsphere solution (3.0 mL / bottle), adiponectin antibody acridinium ester marker (6.0 mL / bottle), sample diluent (13.0 mL / bottle), pre-excitation solution (1.0 mL / bottle), excitation solution (1.0 mL / bottle) and calibrator (0.5 mL / tube, 3 tubes).

[0038] The adiponectin antibody magnetic microsphere solution described in this example was prepared by the following method:

[0039] (1) 10 g of methyl orthosilicate, 1.2 g of active end-capping agent 1,3-bis(aminopropyl)tetramethyldisiloxane and 20 g of magnetic ferrosoferric oxide nanoparticles were added to a mixed solvent of 60 g of water and ethanol (V 水 :V 乙醇 =5:5) and stirred to mix evenly, heated to 80±5°C under nitrogen protection, and then added with hydrochloric acid catalyst solution for stirring reaction for 2h, and the stirring speed was maintained at 200-250r / min during the reaction process. After the reaction was completed, it was filtered, washed with water and ethanol in sequence, and dried with air flow to obtain functionalized MQ silicone resin-coated magnetic microspheres.

[0040] (2) The functionalized MQ silicone resin-coated magnetic microspheres obtained in step (1) were mixed and incubated with a 0.5 mg / mL streptavidin solution on a shaker at room temperature to obtain streptavidin-coated magnetic microspheres.

[0041] (3) Biotin (commercial activated product) was mixed with adiponectin antibody, and the molar ratio of activated biotin to adiponectin antibody was set to 1:2. The mixture was reacted at room temperature and centrifuged. The supernatant was dialyzed in PBS buffer at 4°C to obtain biotinylated adiponectin antibody.

[0042] (4) The streptavidin-coated magnetic microspheres obtained in step (2) and the biotinylated adiponectin antibody obtained in step (3) were mixed and incubated at a mass ratio of 100:6 on a shaker at room temperature for 2 hours to obtain an adiponectin antibody magnetic microsphere solution. The obtained adiponectin antibody magnetic microsphere solution was diluted to 2 mg / mL with a phosphate buffer having a pH of 7.4 before use.

[0043] The adiponectin antibody fluorescein marker described in this example is an adiponectin antibody acridinium ester marker, which can be obtained by mixing and incubating acridinium ester and adiponectin antibody in a phosphate buffer solution.

[0044] The sample diluent described in this embodiment is a phosphate buffer with a pH of 7.4, containing 1% bovine serum albumin, 0.5% casein, 0.1% Triton X-100, and 0.1% sodium azide preservative.

[0045] In this embodiment, the pre-stimulation solution is a NaOH solution with a concentration of 0.1 mol / L, and the stimulation solution is a H2O2 solution with a concentration of 0.1 mol / L.

[0046] The calibrator described in this example is a homemade phosphate buffer containing adiponectin recombinant antigen (without human-derived substances), protein stabilizer BSA and preservative sodium azide.

[0047] The adiponectin assay kit of this embodiment is used to perform detection using a double antibody sandwich immunoassay using magnetic microparticle chemiluminescence technology (using a fully automatic chemiluminescence immunoassay analyzer, and calibrating the measurement system with a working calibrator before detection to obtain a calibration curve). The specific detection steps are as follows:

[0048] (1) First step of incubation: After adding the sample and sample diluent and automatically diluting, take the pre-diluted sample and adiponectin antibody magnetic microsphere solution. The specific adiponectin in the sample reacts with the adiponectin antibody 1 coated on the magnetic microparticles to form an antigen-antibody 1 complex.

[0049] (2) Cleaning: Under the action of the magnetic field, the magnetic particles are adsorbed to the wall of the reaction tube, and the unbound substances are washed away by the cleaning solution.

[0050] (3) Second incubation step: Add adiponectin antibody 2-acridinium ester label to react with the antigen-antibody 1 complex formed in the first step to form an antibody 1-antigen-antibody 2 complex.

[0051] (4) Clean again.

[0052] (5) Excitation and reading: Pre-excitation solution and excitation solution are added to the reaction complex, and the chemiluminescent reaction is measured by the relative luminescence intensity.

[0053] (6) The adiponectin content in the sample is positively correlated with the relative luminescence intensity measured by the optical system of the measuring instrument.

[0054] (7) The test results are determined by the calibration curve.

[0055] 1. Linear range test

[0056] The kit of this embodiment was used to test five different concentrations of adiponectin corporate reference products within the linear range (0.05 mg / L to 60.00 mg / L). Each corporate reference product was tested three times and the average value of each concentration was calculated. According to formulas (1), (2), and (3), the linear equation y=a+bx (y: test value of the reagent, x: actual value of the corporate reference product) and the correlation coefficient r were calculated.

[0057]

[0058]

[0059]

[0060] Where:

[0061] b-slope of the regression line;

[0062] |a| - absolute value of the intercept of the regression line;

[0063] r - correlation coefficient;

[0064] X i - Actual value of the enterprise reference product;

[0065] Y i - The mean of three repeated measurements of the company's reference product;

[0066] i-1, 2, 3, ..., n;

[0067] n - number of samples measured.

[0068] The results showed that within the detection range of the kit, 0.05 to 60.00 mg / L, the linear correlation coefficient r was 0.9992.

[0069] 2. Detection limit test

[0070] Five samples with a concentration close to 0.05 mg / L were selected for testing. Each sample was tested 5 times, and the test results were sorted by size. The results showed that the test results were all <3.0 mg / L, and there was no test result below the blank limit (0.03 mg / L). This shows that the detection limit of the kit is not greater than 0.05 mg / L.

[0071] 3. Repeatability Test

[0072] Under repeatability conditions, the normal physiological value (0.50 mg / L) and the abnormal physiological value (20.6 mg / L) of the adiponectin enterprise reference product were measured using the kit, and the measurements were repeated 10 times, and the average values ​​of the measured values ​​were calculated respectively ( X ) and standard deviation (S). The coefficient of variation (CV) was calculated according to formula (4).

[0073] CV=S / X *100%......................................(4);

[0074] The results of repeated measurements showed that the coefficient of variation (CV) of the normal physiological value reference was 1.6%, and the coefficient of variation (CV) of the abnormal physiological value reference was 0.82%.

[0075] 4. Accuracy Test

[0076] The kit of this embodiment was used to measure three different concentrations of adiponectin corporate reference products within the linear range, namely high (60.00 mg / L), medium (10.00 mg / L), and low (0.05 mg / L). Each corporate reference product was measured three times, the mean of the sample measurement results was calculated, and the relative deviation (Bias%) was calculated according to formula (5).

[0077] Bias%=( X -X) / X*100%................................(5);

[0078] X - Mean value of sample measurement results;

[0079] X-theoretical value of the measured sample.

[0080] The results showed that the relative deviations of high, medium and low concentrations of corporate reference products were -2.3%, 0.95% and -1.8% respectively.

[0081] V. Calibrator Measurement Accuracy Test

[0082] The kit calibrator was used as the sample for testing. After repeated measurement for 3 times, the absolute deviation (M) of CAL1 (labeled value 0.50 mg / L) was calculated according to formula (6). D ), and the relative deviation (B) of CAL2 (labeled value 5.00 mg / L) and CAL3 (labeled value 50.00 mg / L) was calculated according to formula (7): i ).

[0083] M D =|Xi-T|........................(6);

[0084] Bi=(Xi-T) / T×100%................................(7);

[0085] Where:

[0086] X i - Determine the concentration value;

[0087] T—labeled value of the matching calibration material.

[0088] The CAL1 calibrator was tested 3 times. DThe results were 0.02mg / L, 0.01mg / L, and 0.01mg / L, respectively; the Bi results of the CAL2 calibrator measured three times were -1.27%, -2.35%, and 0.66%, respectively; the Bi results of the CAL3 calibrator measured three times were -3.46%, 1.96%, and -2.60%, respectively.

[0089] It can be seen from the above results that the adiponectin determination kit of the present invention has a good linear range, a low detection limit, good repeatability and a high accuracy.

[0090] Comparative Example 1

[0091] Compared with Example 1, this comparative example uses amino-modified silica (coated) magnetic microspheres to replace the functionalized MQ silicone resin coated magnetic microspheres. The adiponectin antibody magnetic microsphere solution is prepared by the following method:

[0092] (1) 20 g of silica magnetic microspheres (commercially available, silica-coated ferroferric oxide magnetic microspheres, average particle size 200 nm) were added to 60 g of a mixed solvent of water and ethanol (V 水 :V 乙醇 =5:5) and stirred to mix evenly, then 1.2g of 3-aminopropyltrimethoxysilane coupling agent was added to stir and react for 2h. After the reaction was completed, it was filtered, washed with water and ethanol in sequence, and dried in air flow to obtain amino-modified silica magnetic microspheres.

[0093] (2) The amino-modified silica magnetic microspheres obtained in step (1) were mixed and incubated with a 0.5 mg / mL streptavidin solution on a shaker at room temperature to obtain streptavidin-coated magnetic microspheres.

[0094] (3) Biotin (commercial activated product) was mixed with adiponectin antibody, and the molar ratio of activated biotin to adiponectin antibody was set to 1:2. The mixture was reacted at room temperature and centrifuged. The supernatant was dialyzed in PBS buffer at 4°C to obtain biotinylated adiponectin antibody.

[0095] (4) The streptavidin-coated magnetic microspheres obtained in step (2) and the biotinylated adiponectin antibody obtained in step (3) were mixed and incubated at a mass ratio of 100:6 on a shaker at room temperature for 2 hours to obtain an adiponectin antibody magnetic microsphere solution. The obtained adiponectin antibody magnetic microsphere solution was diluted to 2 mg / mL with a phosphate buffer having a pH of 7.4 before use.

[0096] The adiponectin antibody magnetic microsphere solution of this comparative example was used to prepare an adiponectin assay kit according to the specifications and reagents of Example 1. The accuracy test was performed according to the method of step 4 in Example 1, and the results showed that the relative deviations of the high, medium and low concentrations of the enterprise reference products were -11.5%, -12.6% and -12.1%, respectively. The calibration product measurement accuracy test was performed according to the method of step 5 in Example 1, and the results showed that the CAL1 calibration product was measured 3 times with M D The results were 0.07mg / L, 0.08mg / L, and 0.08mg / L, respectively; the Bi results of the CAL2 calibrator measured three times were -10.37%, -12.84%, and -11.52%, respectively; the Bi results of the CAL3 calibrator measured three times were -12.75%, -14.56%, and -13.90%, respectively.

[0097] By comparing the accuracy test results of this comparative example with those of Example 1, it can be seen that the kit of the present invention can significantly improve the detection accuracy by coating magnetic microspheres with functionalized MQ silicone resin. The reason is that the MQ silicone resin coating has a unique spherical structure with a tight inner layer and loose functional groups in the outer layer. Its inner layer is tightly combined with inorganic magnetic nanoparticles, and the coating effect is good; the functional groups are evenly distributed in the outermost layer of the microspheres, and the binding efficiency with biologically active substances such as antibodies and antigens is high, which can significantly improve the effect of magnetic separation, thereby improving the detection accuracy. However, the controllability of surface modification using aminosilane coupling agents is poor, and the self-polymerization cross-linking reaction itself is easy to produce, which will cause the active groups to be embedded and reduce the surface modification effect, thereby leading to a decrease in activity and reactivity, so the binding with biologically active substances is reduced, and the effect of magnetic separation is reduced, resulting in a low detection result.

[0098] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing adiponectin antibody magnetic microspheres, characterized in that: The method comprises the following preparation steps: (1) adding silicate, active end-capping agent and magnetic nanoparticles into a solvent and stirring to mix evenly, heating to 60-90°C under inert atmosphere, then dropping an acid catalyst solution to react with stirring, filtering, washing and drying after the reaction is completed to obtain functionalized MQ silicone resin-coated magnetic microspheres; (2) mixing the functionalized MQ silicone resin-coated magnetic microspheres obtained in step (1) with a streptavidin solution and incubating them to obtain streptavidin-coated magnetic microspheres; (3) mixing biotin and adiponectin antibody to obtain biotinylated adiponectin antibody; (4) mixing and incubating the streptavidin-coated magnetic microspheres obtained in step (2) with the biotinylated adiponectin antibody obtained in step (3) to obtain adiponectin antibody magnetic microspheres; In step (1), the silicate is at least one of methyl orthosilicate and ethyl orthosilicate; the active end-capping agent is at least one of 1,3-bis(aminopropyl)tetramethyldisiloxane, 1,3-bis(3-carboxypropyl)tetramethyldisiloxane, 1,3-bis(3-hydroxypropyl)tetramethyldisiloxane, 1,3-bis(3-glycidylpropyl)tetramethyldisiloxane, 1,3-divinyltetramethyldisiloxane, and tetramethyldisiloxane; the magnetic nanoparticles are selected from magnetic ferrosoferric oxide nanoparticles; and the acid catalyst solution is selected from hydrochloric acid solution or sulfuric acid solution; The mass ratio of the silicate, active end-capping agent and magnetic nanoparticles added is 10:0.5-2:10-40.

2. The method for preparing adiponectin antibody magnetic microspheres according to claim 1, characterized in that: The solvent in step (1) is a mixed solvent of water and a small molecule alcohol, and the volume ratio of the water to the small molecule alcohol is 4-7:6-3; the stirring reaction time is 1-4 hours.

3. The method for preparing adiponectin antibody magnetic microspheres according to claim 1, characterized in that: The concentration of the streptavidin solution in step (2) is 0.1-1 mg / mL; the molar ratio of the biotin and the adiponectin antibody in step (3) is 1:1-5; the adiponectin antibody in step (3) refers to an anti-human adiponectin monoclonal antibody or an anti-human adiponectin polyclonal antibody; the mass ratio of the streptavidin-coated magnetic microspheres and the biotinylated adiponectin antibody mixed and incubated in step (4) is 100:5-10.

4. Adiponectin antibody magnetic microspheres, characterized in that: It is prepared by the method according to any one of claims 1 to 3.

5. A kit for determining adiponectin, characterized in that: The method comprises the adiponectin antibody magnetic microsphere solution as claimed in claim 4, an adiponectin antibody fluorescent marker and a sample diluent.

6. The adiponectin assay kit according to claim 5, characterized in that: The adiponectin antibody fluorescein marker is an adiponectin antibody acridinium ester marker; the sample diluent is a phosphate buffer containing bovine serum albumin, casein, a surfactant and a preservative.

7. The adiponectin assay kit according to claim 5, characterized in that: The adiponectin assay kit also includes a pre-stimulation solution and a stimulation solution; the pre-stimulation solution is a NaOH solution with a concentration of 0.05-0.25 mol / L, and the stimulation solution is a H2O2 solution with a concentration of 0.05-0.2 mol / L.

8. The adiponectin assay kit according to claim 5, characterized in that: The adiponectin assay kit also includes a calibrator; the calibrator is a phosphate buffer containing adiponectin recombinant antigen, a protein stabilizer and a preservative.

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