Adiponectin ADP monoclonal antibody and application

Abstract

The invention relates to the technical field of monoclonal antibodies, in particular to an adiponectin ADP monoclonal antibody and application, the heavy chain sequence of the adiponectin ADP monoclonal antibody is shown as SEQ ID NO: 1, and the light chain sequence of the adiponectin ADP monoclonal antibody is shown as SEQ ID NO: 2. The adiponectin ADP monoclonal antibody disclosed by the invention has excellent specificity and sensitivity, can provide accurate and reliable reagent guarantee for related detection scenes, can be applied to a plurality of detection fields such as biochemical immunoturbidimetry, immunochromatography and ELISA (Enzyme-Linked Immunosorbent Assay), and realizes rapid, efficient and high-sensitivity clinical detection on an ADP antigen in serum.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to an adiponectin ADP monoclonal antibody and its applications. Background Technology

[0002] Adiponectin (ADP) is an adipokine secreted by adipocytes that plays a key role in regulating glucose metabolism, fatty acid oxidation, and insulin sensitivity. It also possesses physiological activities such as anti-diabetic, anti-atherosclerotic, and anti-inflammatory effects. Adiponectin levels are negatively correlated with obesity, metabolic syndrome, type 2 diabetes, and cardiovascular disease, making it an important biomarker for assessing the risk, progression, and prognosis of these diseases.

[0003] Currently, the main methods for detecting adiponectin are enzyme-linked immunosorbent assay (ELISA) and latex-enhanced immunoturbidimetry, which rely on highly specific and high-affinity adiponectin antibodies. While various adiponectin antibodies and antibody pairs exist in existing technologies, they generally suffer from the following drawbacks: some antibodies lack specificity and cross-react with structural analogs such as leptin and resistin; some antibodies have low affinity, resulting in limited detection sensitivity and inability to accurately detect low-concentration samples; antibody pair pairing performance is poor, leading to poor repeatability and stability of test results, and significant batch-to-batch variability; furthermore, existing antibodies are easily inactivated in kits, requiring complex stabilizer systems to maintain activity, increasing detection costs.

[0004] Therefore, developing an adiponectin ADP antibody with high specificity, high affinity, and excellent stability, and applying it to in vitro diagnostic test kits, is of great significance for improving the accuracy and reliability of adiponectin detection in serum or plasma. Summary of the Invention

[0005] The purpose of this invention is to provide an adiponectin ADP monoclonal antibody with high specificity and sensitivity and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: An adiponectin ADP monoclonal antibody, the heavy chain sequence of which is shown in SEQ ID NO:1 and the light chain sequence of which is shown in SEQ ID NO:2.

[0007] The adiponectin (ADP) monoclonal antibody described in this invention can be used to prepare a latex immunoturbidimetric assay kit. The kit includes reagent R1 and reagent R2. The reagent R1 is 50mM pH 6.5 MES buffer, Tween 80 1g / L, PEG20000 2g / L, BSA 0.9%, and Proclin 300 0.1% v / v; The preparation method of reagent R2 is as follows: Take 0.125 mL of PS microspheres, add 1 mL of 25 mM pH 6.5 MES buffer and mix, then add 50 μL of 0.01 g / mL EDC solution, mix and stir at room temperature for 15 min; centrifuge at 12000 rpm for 10 min, add 2 mL of 25 mM pH 6.5 MES buffer to resuspend, and sonicate at 30% power for 1 min; add 0.3 mg of ADP mAb to the reaction system and react at room temperature for 1 h; add 20 μL of blocking agent to the reaction system and react at room temperature for 1 h; centrifuge at 7000 rpm for 5 min, resuspend in 3 mL of pH 7.0 PBS solution, sonicate at 30% power for 3 min, and store at 2-8℃ for later use.

[0008] The adiponectin (ADP) monoclonal antibody described in this invention can be used to prepare an immunochromatographic assay kit. The preparation method is as follows: (1) Labeling antibody: Take 0.05 mg of colored microspheres into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 50 μL of 10 mg / mL EDC, vortex to mix, and incubate at room temperature for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres in 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of ADP mAb, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% w / v BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use; (2) Preparation of nitrocellulose membrane: ADP mAb was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as a detection line, and goat anti-mouse IgG antibody at a concentration of 1 mg / mL was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as a quality control line. The membrane was dried overnight at 45°C. (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads; (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0009] The adiponectin ADP monoclonal antibody described in this invention can also be used to prepare ELISA detection kits.

[0010] Compared with the prior art, the beneficial effects of the present invention are: (1) The adiponectin ADP monoclonal antibody of the present invention has excellent specificity and sensitivity, and can provide accurate and reliable reagent support for relevant detection scenarios.

[0011] (2) The adiponectin ADP monoclonal antibody of the present invention can be used in multiple detection fields such as biochemical immunoturbidimetry, immunochromatography and ELISA to achieve rapid, efficient and highly sensitive clinical detection of ADP antigen in serum. Attached Figure Description

[0012] Figure 1 The results of SDS-PAGE analysis of purified adiponectin ADP monoclonal antibody are shown.

[0013] Figure 2 This is the SEC-HPLC chromatogram of the purified adiponectin ADP monoclonal antibody.

[0014] Figure 3 The results are from the calibrator test of the latex immunoturbidimetric ADP assay kit. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Preparation of Adiponectin (ADP) Monoclonal Antibody (1) Immunized animals The immunization regimen is selected based on the characteristics of the antigen. For soluble antigens with weak immunogenicity, adjuvants are generally added. Commonly used adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and adjuvant should be mixed in equal volumes and ground into a water-in-oil emulsion.

[0017] The initial immunization was performed by subcutaneous injection of 50 μg antigen with Freund's complete adjuvant at multiple sites; the second immunization was performed 3 weeks later (same dose of antigen with Freund's incomplete adjuvant); the third immunization was performed by intraperitoneal injection without adjuvant, and the titer was tested 7 days later; the final booster immunization was performed (50 μg antigen intraperitoneal injection), and spleen cells were harvested for fusion 3 days later.

[0018] (2) Cell fusion Myeloma cell suspension: Select a suitable myeloma cell line, isolate myeloma cells from tumor cells growing in vivo, and prepare a cell suspension.

[0019] Spleen cell suspension: Mice that received a booster 3 days prior were subjected to orbital bloodletting, and the serum was separated and frozen for later use. The mice were euthanized and immersed in 75% alcohol for 3-5 minutes. The spleen was aseptically removed and washed in a petri dish containing 5 mL of incomplete culture medium. The surrounding connective tissue was removed, and the spleen was transferred to a steel mesh in another petri dish containing 5 mL of incomplete culture medium. It was first cut into 3-5 small pieces with scissors, then ground with a syringe core. The spleen cell suspension was transferred to a 50 mL centrifuge tube, 50 mL of incomplete culture medium was added, and the mixture was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the mixture was washed and centrifuged again using the same method. The precipitated cells were then resuspended in 10 mL of incomplete culture medium, and the viable cell count was recorded. 0.5-2 × 10⁶ cells were obtained from one mouse. 8 One spleen cell.

[0020] Feeder Cells: After euthanizing, disinfecting, and fixing the mice, use sterile scissors to lift the abdominal skin from the posterior abdomen to expose the peritoneum. Disinfect the peritoneum with an alcohol swab. Inject 10 mL of incomplete culture medium into the peritoneal cavity using a syringe, taking care to avoid perforation of the intestine. Hold the syringe firmly with your right hand, leaving the needle in the peritoneal cavity, and gently massage the abdomen with an alcohol swab in your left hand for 1 minute. Then aspirate the injected culture medium. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the pelleted cells in 5 mL of HAT medium. Based on the cell count, add more HAT medium to bring the cell concentration to 2 × 10⁶ cells / mL. 5 / mL, for later use.

[0021] Cell fusion: Mix the prepared myeloma cells and mouse spleen cells at a ratio of 1:5, add 20 mL of PRMI-1640 solution; centrifuge at 1000 r / min for 10 min, discard the supernatant, and aspirate as much supernatant as possible; gently tap the bottom of the centrifuge tube with your finger to disperse the precipitated cells, and place the centrifuge tube in a 37℃ water bath; slowly add 1 mL of 50% PEG pre-warmed at 37℃ into the centrifuge tube over about 45 seconds, stirring gently while adding, and let stand at 37℃ for 5 min; add 20 mL of incomplete culture medium (pre-warmed at 37℃) over 5 min, adding 1 mL in the first minute, 2 mL in the second minute, 5 mL in the third minute, and 6 mL each in the fourth and fifth minutes, while gently rotating the centrifuge tube. The medium should be added along the tube wall, not directly onto the precipitated cells, to prevent dislodging the newly fused cells. Then add PRMI-1640 solution to 50 mL to terminate the PEG effect; centrifuge at 800 r / min for 10 min, discard the supernatant, gently suspend the precipitated cells in the required volume of HAT culture medium, and seed 0.10-0.15 mL per well in a 96-well culture plate. After seeding, place the culture plate in a 37℃ 5% CO2 incubator for culture.

[0022] (3) Selective culture Five days after inoculating 96-well plates, replace half of the medium with HAT medium. After 7-10 days, replace the HAT medium with HT medium. After the 14th day, ordinary complete medium can be used.

[0023] (4) Screening and cloning of hybridoma-positive clones Coating: The antigen was coated with Na2CO3-NaHCO3 buffer at pH 9.6. 20 μg of antigen was added to each microplate, and the coating volume per well was 50 μL. Coating was carried out at 37°C for 2 h.

[0024] Blocking: Discard the antigen, blot dry the liquid in the wells of the ELISA plate, and block with 1% BSA at 37°C for 2 hours.

[0025] Primary antibody: After blocking, discard the liquid in the well, pat dry, add 100 μL of the supernatant of the cells to be tested to each well, and incubate at 37°C for 1 h.

[0026] Secondary antibody: After incubation with primary antibody, discard the liquid in the wells, blot dry, and wash three times with PBST buffer (pH 7.4) for 5 min each time. Dilute the secondary antibody according to the instructions, add 100 μL to each well, and incubate at 37°C for 1 h.

[0027] Color development: After incubation with the secondary antibody, discard the liquid in the wells, pat dry, add the colorimetric reagent, and stop the reaction with the stop solution when the color is the deepest. Read the OD value of each well with a microplate reader, select the positive wells, expand the culture and freeze them.

[0028] (5) Purification of monoclonal antibodies Monoclonal antibodies were purified using an affinity purification method. Staphylococcal A protein was cross-linked with a carrier, and an affinity chromatography column was prepared to bind the antibody before elution. The antibody was then concentrated to 5 mg / mL. The heavy chain sequence of the purified adiponectin (ADP) monoclonal antibody is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2.

[0029] Example 2 The purity of ADP mAb antibody was analyzed by SDS-PAEG electrophoresis and SEC-HPLC, respectively. The results are as follows: Figure 1 and Figure 2 As shown.

[0030] SDS-PAEG electrophoresis experimental procedure: A. Prepare the separating adhesive and the concentrating adhesive, pour them into the adhesive in sequence, and then remove the comb after solidification.

[0031] B. Mix the protein sample with SDS loading buffer at a ratio of 4:1, and denature in a boiling water bath for 5-10 minutes before cooling.

[0032] C. Load the gel into the electrophoresis tank, fill with buffer and add the sample. Electrophore at a constant voltage of 80V until the indicator enters the separating gel, then adjust to 120V until the indicator is close to the bottom of the gel.

[0033] D. Remove the gel, stain with Coomassie Brilliant Blue staining solution for at least 30 minutes with shaking, and change the destaining solution several times with shaking until the bands are clear and the background is transparent.

[0034] SEC-HPLC experimental procedure: A. Equilibrate the column with phosphate buffer (mobile phase) until the baseline is stable.

[0035] B. The sample is filtered through a 0.22 μm filter membrane to avoid particulate contamination of the chromatographic column.

[0036] C. Set the HPLC parameters (flow rate 0.5-1.0 mL / min, column temperature 30℃), and inject 25 μL of sample after the system stabilizes.

[0037] D. After the operation is complete, flush the column with the mobile phase and seal with pure solvent. During the operation, it is necessary to degas the mobile phase, prevent sudden increases in column pressure, and avoid air bubbles in the system.

[0038] Example 3: Preparation of ADP Detection Kit (Latex Immunoturbidimetric Assay) using ADP Monoclonal Antibody from Example 1 Reagent R1: 50mM pH 6.5 MES buffer, Tween 80 1g / L, PEG20000 2g / L, BSA 0.9w%, Proclin 300 0.1% (v / v).

[0039] Preparation of reagent R2: Take 0.125 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102025), add 1 mL of 25 mM pH 6.5 MES buffer, mix, then add 50 μL of 0.01 g / mL EDC solution (freshly prepared), mix and stir at room temperature for 15 min; centrifuge at 12000 rpm for 10 min, resuspend in 2 mL of 25 mM pH 6.5 MES buffer, sonicate at 30% power for 1 min; add 0.3 mg of ADP mAb prepared in Example 1 to the reaction system, react at room temperature for 1 h; add 20 μL of blocking agent (blocking agent is 0.9% BSA and 0.2% glycine aqueous solution) to the reaction system, react at room temperature for 1 h; centrifuge at 7000 rpm for 5 min, resuspend in 3 mL of pH 7.0 PBS solution, sonicate at 30% power for 3 min, and store at 2-8℃ for later use.

[0040] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0041] Parameters: Sample volume 3μL, reagent R1 160μL, reagent R2 40μL, fitting method Spline, main wavelength 700nm, secondary wavelength none.

[0042] Example 4: Preparation of ADP mAb monoclonal antibody from Example 1 into an ADP detection kit (color latex immunochromatography). (1) Labeling antibody: Take 0.05 mg of colored microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD202002) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 50 μL of 10 mg / mLEDC, vortex to mix, and incubate at room temperature for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of ADP mAb from Example 1, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres with 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use.

[0043] (2) Preparation of nitrocellulose membrane: ADP mAb from Example 1 was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as a detection line, and goat anti-mouse IgG antibody at a concentration of 1 mg / mL was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as a quality control line. The membrane was dried overnight at 45°C.

[0044] (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads.

[0045] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0046] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0047] Comparative Example 1: An ADP detection kit (latex immunoturbidimetric assay) was prepared using HyTest MAb Adn23 monoclonal antibody. Reagent R1: 50mM pH 6.5 MES buffer, Tween 80 1g / L, PEG20000 2g / L, BSA 0.9w%, Proclin 300 0.1% (v / v).

[0048] Preparation of reagent R2: Take 0.125 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102025), add 1 mL of 25 mM pH 6.5 MES buffer, mix, then add 50 μL of 0.01 g / mL EDC solution (freshly prepared), mix and stir at room temperature for 15 min; centrifuge at 12000 rpm for 10 min, resuspend in 2 mL of 25 mM pH 6.5 MES buffer, sonicate at 30% power for 1 min; add 0.3 mg of MAb Adn23 (HyTest Biotechnology Co., Ltd., catalog number: 2AN6) to the reaction system, react at room temperature for 1 h; add 20 μL of blocking agent (blocking agent is 0.9% BSA and 0.2% glycine aqueous solution) to the reaction system, react at room temperature for 1 h; centrifuge at 7000 rpm for 5 min, resuspend in 3 mL of pH 7.0 PBS solution, sonicate at 30% power for 3 min, and store at 2-8℃ for later use.

[0049] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0050] Parameters: Sample volume 3μL, reagent R1 160μL, reagent R2 40μL, fitting method Spline, main wavelength 700nm, secondary wavelength none.

[0051] Comparative Example 2: An ADP detection kit (color latex immunochromatography) was prepared using HyTest MAb Adn23 monoclonal antibody. (1) Antibody labeling: Take 0.05 mg of colored microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD202002) into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 50 μL of 10 mg / mLEDC, vortex to mix, and incubate at room temperature for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, and resuspend the microspheres with 1 mL of 25 mM pH 6.5 MES buffer, repeating 3 times; add 0.05 mg of MAb Adn23 (HyTest, catalog number: 2AN6) from Example 1, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% (w / v) BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, and resuspend the microspheres with 1 mL of 50 mM pH 7.4 MES buffer. Resuspend the microspheres in PBS, repeat 3 times, and store at 2-8℃ for later use.

[0052] (2) Preparation of nitrocellulose membrane: MAb Adn23 (HyTest Biotechnology Co., Ltd., catalog number: 2AN6) was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as the detection line, and goat anti-mouse IgG antibody at a concentration of 1 mg / mL was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as the quality control line. The membrane was dried overnight at 45°C.

[0053] (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads.

[0054] (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad (glass cellulose membrane) on the conjugate pad, and attach the absorbent pad to one end of the control line.

[0055] Figure 3 The calibrator test results of the latex immunoturbidimetric ADP detection kit are compared with those of Example 3 and Comparative Example 1. 2 The values ​​were 0.9937 and 0.9903, respectively, indicating a very high linear fit between the calibrator concentration and absorbance, effectively ensuring the accuracy of the kit. Furthermore, the absorbance change for the same concentration variation in Example 3 was more significant, indicating that the ADP kit prepared in Example 3 has better detection sensitivity than Comparative Example 1.

[0056] Table 1 shows the calibrator test results of the color latex immunochromatographic ADP detection kit. In the blank group, the detection line (T) signals of Comparative Example 2 and Example 4 were at extremely low levels, indicating that the kit has low background interference and good basic stability. As the concentration of the calibrator increased, the detection line (T) signals of both showed an upward trend. At the same concentration, the T signal of Example 4 was significantly higher than that of Comparative Example 2, indicating that the color was more obvious. Therefore, the detection sensitivity of Example 4 was better than that of Comparative Example 2.

[0057] Table 1. ADP calibrator testing (color latex immunochromatography) Note: The signal value is obtained by the card reader. The higher the value, the more obvious the color display.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An adiponectin (ADP) monoclonal antibody, characterized in that: Its heavy chain sequence is shown in SEQ ID NO:1, and its light chain sequence is shown in SEQ ID NO:

2.

2. The use of the adiponectin ADP monoclonal antibody as described in claim 1 in the preparation of a latex immunoturbidimetric assay kit.

3. The application of the adiponectin ADP monoclonal antibody according to claim 2 in the preparation of a latex immunoturbidimetric assay kit, characterized in that: The kit includes reagent R1 and reagent R2; The reagent R1 is 50mM pH 6.5 MES buffer, Tween 80 1g / L, PEG20000 2g / L, BSA 0.9%, and Proclin 300 0.1% v / v; The preparation method of reagent R2 is as follows: Take 0.125 mL of PS microspheres, add 1 mL of 25 mM pH 6.5 MES buffer and mix, then add 50 μL of 0.01 g / mL EDC solution and mix and stir at room temperature for 15 min; centrifuge at 12000 rpm for 10 min, add 2 mL of 25 mM pH 6.5 MES buffer to resuspend, and sonicate at 30% power for 1 min; add 0.3 mg of ADP mAb to the reaction system and react at room temperature for 1 h; add 20 μL of blocking agent to the reaction system and react at room temperature for 1 h; centrifuge at 7000 rpm for 5 min, resuspend in 3 mL of pH 7.0 PBS solution, sonicate at 30% power for 3 min, and store at 2-8℃ for later use.

4. The use of the adiponectin ADP monoclonal antibody as described in claim 1 in the preparation of an immunochromatographic detection kit.

5. The application of the adiponectin ADP monoclonal antibody according to claim 4 in the preparation of an immunochromatographic detection kit, characterized in that, The preparation method of the reagent kit is as follows: (1) Labeling antibody: Take 0.05 mg of colored microspheres into a centrifuge tube, add 1 mL of 25 mM pH 6.5 MES buffer, and vortex to mix; add 50 μL of 10 mg / mL EDC, vortex to mix, and incubate at room temperature for 30 min; centrifuge the activated microspheres at 15000 rpm for 20 min, remove the supernatant, resuspend the microspheres in 1 mL of 25 mM pH 6.5 MES buffer, and repeat 3 times; add 0.05 mg of ADP mAb, vortex to mix, and incubate at room temperature for 2 h; add 100 μL of 10% w / v BSA, and incubate at room temperature for 30 min; centrifuge at 15000 rpm for 15 min, remove the supernatant, resuspend the microspheres in 1 mL of 50 mM pH 7.4 PBS, repeat 3 times, and store at 2-8℃ for later use; (2) Preparation of nitrocellulose membrane: ADP mAb was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as a detection line, and goat anti-mouse IgG antibody at a concentration of 1 mg / mL was applied to the nitrocellulose membrane at a coating amount of 1 μL / cm as a quality control line. The membrane was dried overnight at 45°C. (3) Preparation of conjugate pads: The antibody complex labeled with colored microspheres was sprayed onto a glass cellulose membrane at a coating amount of 6 μL / cm and dried overnight at 45°C to prepare conjugate pads; (4) Test strip assembly: attach the coated nitrocellulose membrane to the support base plate, attach the conjugate pad to one end of the marking line, place the sample pad on the conjugate pad, and attach the absorbent pad to one end of the control line.

6. The use of the adiponectin ADP monoclonal antibody as described in claim 1 in the preparation of an ELISA detection kit.

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