A pair of adiponectin antibodies and a latex-enhanced immunoturbidimetric assay kit for adiponectin determination

Abstract

The present invention provides a pair of adiponectin antibodies, including ADPN antibody I and ADPN antibody II; ADPN antibody I includes a light chain and a heavy chain, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; ADPN antibody II includes a light chain and a heavy chain, the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The present invention also provides a kit for determining adiponectin by latex-enhanced immunoturbidimetry, including the above-mentioned adiponectin antibodies. The adiponectin antibodies of the present invention can bind to ADPN in serum, and after binding, they can increase the activity of ADPN, thereby significantly increasing the sensitivity, accuracy, stability and repeatability of the reagent in the ADPN latex immunoturbidimetry detection; at the same time, the present invention adds glycerol, block polyether F68 and Surfynol 465 as stabilizers to the kit to further improve stability.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a pair of adiponectin antibodies and a kit for determining adiponectin by latex-enhanced immunoturbidimetry based on the antibodies. Background Art

[0002] Adiponectin (ADPN), an endogenous bioactive peptide or protein secreted by adipocytes, is an insulin-sensitizing hormone. Adiponectin testing is primarily used to predict diabetes risk and screen high-risk individuals for the condition, helping to detect diabetes early and reduce morbidity and complications.

[0003] Numerous studies have demonstrated an inverse relationship between adiponectin concentration and insulin resistance. Adiponectin can increase insulin levels, reflecting insulin sensitivity. Therefore, adiponectin can be used as a predictor of diabetes risk. Adiponectin can be used to screen individuals at high risk for diabetes, even though their blood sugar levels remain normal but their physiological metabolism is abnormal. Furthermore, adiponectin levels in the blood are stable, and its detection is unaffected by factors such as food intake, mood swings, lack of sleep, and changes in dietary habits.

[0004] In addition to blood sugar metabolism, adiponectin also participates in lipid metabolism, inducing a decrease in circulating lipid levels and exerting a lipid-lowering effect in the body. Obesity is considered a risk factor for various cardiovascular diseases and malignancies. Adiponectin can inhibit the formation of lipid plaques in arteries and prevent the growth and proliferation of cancer cells. Therefore, adiponectin testing can also be used to assess cardiovascular disease risk and detect cancer early.

[0005] However, the main adiponectin assays currently available on the market are enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assays. ELISA is low-cost and requires simple instrumentation, but its results are less stable and reproducible, and the assay is time-consuming. Chemiluminescence assays, on the other hand, require expensive instruments and often require closed channels, making them unsuitable for use in hospitals below the secondary level.

[0006] Latex-enhanced immunoturbidimetry (PETIA) utilizes the specific binding of antigens and antibodies. Antibodies are cross-linked or physically adsorbed on the surface of nanoscale latex microspheres. After the cross-linked antibody microspheres in the reagent reaction solution bind to the antigens in the sample, they rapidly aggregate within a short period of time, forming turbidity, thereby changing the light scattering or light transmittance of the reaction solution. This method can reflect the concentration of the antigen being tested within the linear range. The advantage of latex-enhanced immunoturbidimetry is that it is easy to operate and results can be obtained in a few minutes. In addition, the simplified operating steps of the fully automated immunoturbidimetry method also avoid many human operating factors and interference from external factors such as reagents and the environment. It has good stability and repeatability, can more accurately reflect the content of the substance being tested, and is not expensive.

[0007] Therefore, it is urgent to develop a new method for adiponectin determination based on latex-enhanced immunoturbidimetry. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a pair of adiponectin antibodies and a latex-enhanced immunoturbidimetric assay kit for adiponectin based on the antibodies. The adiponectin antibodies are used in combination with the corresponding latex-enhanced immunoturbidimetric assay kit for the determination of ADPN, which is not only simple to operate and has a fast detection speed, but also has the advantages of good stability, repeatability, sensitivity and accuracy.

[0009] The present invention adopts the following technical solutions to solve the above technical problems:

[0010] A pair of adiponectin antibodies, including ADPN antibody I and ADPN antibody II; the ADPN antibody I includes a light chain and a heavy chain, and the corresponding amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the ADPN antibody II includes a light chain and a heavy chain, and the corresponding amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] As one of the preferred embodiments of the present invention, the ADPN antibody I and ADPN antibody II can bind to adiponectin in serum and, after binding, increase the activity of adiponectin.

[0012] A latex-enhanced immunoturbidimetric assay kit for adiponectin comprises the adiponectin antibodies, namely, ADPN antibody I and ADPN antibody II.

[0013] As one of the preferred embodiments of the present invention, it specifically includes R1 reagent and R2 reagent;

[0014] The R1 reagent includes: Tris buffer 10-100 mmol / L, sodium chloride 10-50 g / L, Proclin 3000 0.5-1.5 g / L, Tween-20 1-5 g / L, PEG 6000 10-30 g / L, pH 7.0-8.0;

[0015] The R2 reagent comprises: phosphate buffer 5-20 mmol / L, glycerol 250-350 mL / L, block polyether F68 0.5-1.5 g / L, Surfynol 465 0.5-1.5 g / L, Proclin 300 0.5-1.5 g / L, ADPN mixed antibody 30-100 mg / L, 150-400 nm latex microsphere particles 0.5%-1.5%, pH 7.0-8.0;

[0016] Wherein, the ADPN mixed antibody is a mixed antibody of ADPN antibody I and ADPN antibody II.

[0017] As one of the preferred embodiments of the present invention, the R1 reagent specifically comprises: Tris buffer 50 mmol / L, sodium chloride 25 g / L, Proclin 300 1 g / L, Tween-20 1.5 g / L, PEG 6000 20 g / L, pH 7.4.

[0018] As one of the preferred embodiments of the present invention, the R2 reagent specifically includes: phosphate buffer 10 mmol / L, glycerol 300 mL / L, block polyether F68 1 g / L, Surfynol 465 1 g / L, Proclin 300 1 g / L, ADPN mixed antibody 60 mg / L, 250 nm latex microsphere particles 0.5%, pH 7.4.

[0019] As one of the preferred embodiments of the present invention, in the ADPN mixed antibody, the mass ratio of ADPN antibody I to ADPN antibody II is 1:1.

[0020] As one of the preferred embodiments of the present invention, in the R2 reagent, the ADPN mixed antibody is coupled to the latex microsphere particles to form latex particles coated with the ADPN antibody.

[0021] As one of the preferred embodiments of the present invention, the method for preparing the latex particles coated with ADPN antibody is as follows:

[0022] ① Add the latex microspheres to the coupling buffer; then, preheat in a constant temperature water bath;

[0023] ② Add activator EDC solution and NHS solution, mix well, and place in a constant temperature stirrer for reaction;

[0024] ③ Add ADPN antibody I and ADPN antibody II, mix well, and place in a constant temperature stirrer for reaction;

[0025] ④ Add blocking solution, mix well, and place in a constant temperature stirrer for reaction;

[0026] ⑤ Place in centrifuge for centrifugation;

[0027] ⑥ Add preservation buffer, place in an ice water bath, place in an ultrasonic cell disruptor, and fully disperse.

[0028] As one of the preferred embodiments of the present invention, a calibrator is also included; the calibrator includes: PBS buffer 5-20 mmol / L, sodium chloride 5-10 g / L, BSA 5-15 g / L, sodium azide 0.5-1.5 g / L, ADPN antigen 0-40 mg / L, pH 7.0-8.0.

[0029] The advantages of the present invention over the prior art are:

[0030] (1) The pair of adiponectin antibodies (ADPN antibody I and ADPN antibody II) designed by the present invention can bind to ADPN in serum, and after binding, they can increase the activity of ADPN, thereby significantly increasing the sensitivity, accuracy, stability and repeatability of the reagent in the ADPN latex immunoturbidimetric assay;

[0031] (2) The kit of the present invention is based on latex-enhanced turbidimetry. After the antigen and antibody react, the absorbance value of the reaction solution is directly measured, and the result can be obtained in a few minutes. The operation is simple, saving time and labor. At the same time, it has a high degree of automation, strong applicability, convenient testing, and low cost, and can be widely used in clinical testing.

[0032] (3) The present invention adds glycerol, block polyether F68 and Surfynol 465 as stabilizers to the kit to further improve the stability of the test. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the standard curve diagram of the control group and experimental group reagents in Test Example 1. DETAILED DESCRIPTION

[0034] The following embodiments of the present invention are described in detail. These embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the scope of protection of the present invention is not limited to the following embodiments. At the same time, the reagents and experimental methods used in the following embodiments are conventional reagents or methods in the art unless otherwise specified, and will not be repeated here.

[0035] Example 1

[0036] The pair of adiponectin antibodies of this embodiment includes ADPN antibody I and ADPN antibody II.

[0037] The ADPN antibody I includes a light chain and a heavy chain, the light chain amino acid sequence is shown in SEQ ID NO.1, and the heavy chain amino acid sequence is shown in SEQ ID NO.2.

[0038] The ADPN antibody II includes a light chain and a heavy chain, the amino acid sequence of the light chain is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.4.

[0039] Example 2

[0040] This embodiment is a preparation of the adiponectin antibody in the above embodiment 1:

[0041] (1) Expression of adiponectin antibody in CHO-K1 suspension expression system

[0042] Cell Thawing: Thaw a vial of CHO cells (approximately 1 x 10 cells) frozen in liquid nitrogen at 37°C in a water bath. Centrifuge at 300g for 5 minutes. Wipe the vial with alcohol wipes and place in a laminar flow hood. Aspirate the supernatant, resuspend in 20 mL of pre-warmed complete CHO medium, and culture in a 125 mL shake flask at 37°C, 5% CO2, and 120-130 rpm.

[0043] Plasmid extraction: Inoculate the DH5a strain containing the pCDNA4.2 vector containing ADPN antibody I / II in advance (gene synthesised by a biological company, the sequence of ADPN antibody I is shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence of ADPN antibody II is shown in SEQ ID NO.3 and SEQ ID NO.4), culture it at 37°C overnight, and extract the plasmid using a commercial plasmid extraction kit.

[0044] Cell transfection: When cell viability is greater than 95%, there are no obvious cell clumping, and the cell density is between 2 and 3 x 10E6, harvest 4 x 10E7 CHO cells and centrifuge at 300g for 5 minutes. Remove the supernatant and resuspend the CHO cells in 400µL of Celetrix commercial electroporation buffer. Add 25µg of plasmid to deplete 2 x 10E7 CHO cells. Electroporate using a Celetrix cell electroporator at 1250V. Allow the cells to recover for 24 hours.

[0045] Cell transfer and fed-batch culture: After cell recovery, transfer the cells to fermentation medium at a density of 0.5x10E6. Culture until Day 5, then start fed-batch culture. Cool down the culture until Day 7.

[0046] Collect supernatant: Culture until Day 15 or when cell viability drops below 60%. Centrifuge the culture supernatant, remove the cell pellet, and retain the supernatant.

[0047] (2) Affinity purification of "adiponectin antibody"

[0048] Affinity column filling: calculate the required commercial nickel filler, load the filler into the column, and wash the loaded filler with PBS equilibration buffer.

[0049] Loading and washing: The cell supernatant collected by centrifugation was loaded at a low flow rate. After loading, 10 column volumes of equilibration buffer were used to wash the column, and 10 column volumes of pre-elution buffer A containing 20 mM imidazole were used to wash the column.

[0050] Elution and dialysis: After washing the filler with the pre-elution solution, elute it with 250mM imidazole-containing Tris elution solution. The eluted protein is dialyzed overnight with 20mM PBS buffer. After dialysis and changing the buffer, ADPN antibodies I and II are packaged and stored.

[0051] Example 3

[0052] The latex-enhanced immunoturbidimetric assay kit for determining adiponectin in this embodiment includes an R1 reagent, an R2 reagent, and a calibrator.

[0053] R1 reagent includes: Tris buffer 10 mmol / L, sodium chloride 10 g / L, Proclin 300 0.5 g / L, Tween-20 1 g / L, PEG 6000 10 g / L, pH 7.0.

[0054] R2 reagent includes: phosphate buffer 5 mmol / L, glycerol 250 mL / L, block polyether F68 0.5 g / L, Surfynol 465 0.5 g / L, Proclin 300 0.5 g / L, ADPN mixed antibody (ADPN antibody I: ADPN antibody II = 1:1) 30 mg / L, 150 nm latex microsphere particles 0.6%, pH 7.0.

[0055] Calibrators: The calibrators include: PBS buffer 5 mmol / L, sodium chloride 5 g / L, BSA 5 g / L, sodium azide 0.5 g / L, ADPN antigen 0-40 mg / L, pH 7.0.

[0056] Preparation method:

[0057] (1) Prepare R1 reagent:

[0058] According to the component content of R1 reagent, each component is mixed in the same container, and after mixing evenly, R1 reagent is prepared.

[0059] (2) Preparation of latex particles coated with ADPN antibody:

[0060] ① Add the latex microspheres to the coupling buffer (20 mmol / L Hepes buffer, pH 7.5); then, preheat in a constant temperature water bath at 37°C for 20 min.

[0061] ② Add activator EDC solution (10 mg / mL) and NHS solution (50 mg / L), mix well, and place in a constant temperature stirrer to react for 10 minutes at 37°C.

[0062] ③ Add ADPN antibody I and ADPN antibody II, mix well, and place in a constant temperature stirrer to react for 60 minutes at 37°C.

[0063] ④ Add blocking solution 1 (sodium caseinate, 45 g / L) and blocking solution 2 (Tween 20, 100 mL / L), mix well, and place in a constant temperature stirrer to react for 24 h at 37°C.

[0064] ⑤ Centrifuge in a high-speed centrifuge at 10,000 rpm for 30 minutes at 4°C.

[0065] ⑥ Add an appropriate amount of preservation buffer (phosphate buffer 5mmol / L, glycerol 250mL / L, block polyether F680.5g / L, Surfynol 465 0.5g / L, Proclin300 0.5g / L), place in an ice water bath, and place in an ultrasonic cell disruptor to fully disperse for 3 minutes at a power of 300W.

[0066] (3) Prepare R2 reagent:

[0067] According to the component contents of the R2 reagent, the latex particles coated with the ADPN antibody and the remaining components are mixed in the same container and mixed evenly to prepare the R2 reagent.

[0068] (4) Preparation of calibration materials:

[0069] According to the component content of the calibrator, the components are mixed in the same container and mixed evenly to prepare 0-40 mg / L ADPN reagent calibrators, preferably ADPN reagent calibrators with concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L.

[0070] Example 4

[0071] The latex-enhanced immunoturbidimetric assay kit for determining adiponectin in this embodiment includes an R1 reagent, an R2 reagent, and a calibrator.

[0072] R1 reagent includes: Tris buffer 50 mmol / L, sodium chloride 25 g / L, Proclin 300 1 g / L, Tween-20 1.5 g / L, PEG 6000 20 g / L, pH 7.4.

[0073] R2 reagent includes: phosphate buffer 10 mmol / L, glycerol 300 mL / L, block polyether F68 1 g / L, Surfynol 465 1 g / L, Proclin 300 1 g / L, ADPN mixed antibody (ADPN antibody I: ADPN antibody II = 1:1) 60 mg / L, 250 nm latex microsphere particles 0.5%, pH 7.4.

[0074] Calibrators: The calibrators include: PBS buffer 10 mmol / L, sodium chloride 8 g / L, BSA 10 g / L, sodium azide 1 g / L, ADPN antigen 0-40 mg / L, pH 7.4.

[0075] Preparation method:

[0076] (1) Prepare R1 reagent:

[0077] According to the component content of R1 reagent, each component is mixed in the same container, and after mixing evenly, R1 reagent is prepared.

[0078] (2) Preparation of latex particles coated with ADPN antibody:

[0079] ① Add the latex microspheres to the coupling buffer (20 mmol / L Hepes buffer, pH 7.5); then, preheat in a constant temperature water bath at 37°C for 20 min.

[0080] ② Add activator EDC solution (10 mg / mL) and NHS solution (50 mg / L), mix well, and place in a constant temperature stirrer to react for 20 minutes at 37°C.

[0081] ③ Add ADPN antibody I and ADPN antibody II, mix well, and place in a constant temperature stirrer to react for 60 minutes at 37°C.

[0082] ④ Add blocking solution 1 (sodium caseinate, 45 g / L) and blocking solution 2 (Tween 20, 100 mL / L), mix well, and place in a constant temperature stirrer to react for 24 h at 37°C.

[0083] ⑤ Centrifuge in a high-speed centrifuge at 15,000 rpm for 20 minutes at 4°C.

[0084] ⑥ Add an appropriate amount of preservation buffer (phosphate buffer 40mmol / L, glycerol 300mL / L, block polyether F681g / L, Surfynol 465 1g / L, Proclin300 1g / L), place in an ice water bath, and place in an ultrasonic cell disruptor to fully disperse for 5 minutes at a power of 250W.

[0085] (3) Prepare R2 reagent:

[0086] According to the component contents of the R2 reagent, the latex particles coated with the ADPN antibody and the remaining components are mixed in the same container and mixed evenly to prepare the R2 reagent.

[0087] (4) Preparation of calibration materials:

[0088] According to the component content of the calibrator, the components are mixed in the same container and mixed evenly to prepare 0-40 mg / L ADPN reagent calibrators, preferably ADPN reagent calibrators with concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L.

[0089] Example 5

[0090] The latex-enhanced immunoturbidimetric assay kit for determining adiponectin in this embodiment includes an R1 reagent, an R2 reagent, and a calibrator.

[0091] R1 reagent includes: Tris buffer 100 mmol / L, sodium chloride 50 g / L, Proclin 300 1.5 g / L, Tween-20 5 g / L, PEG 6000 30 g / L, pH 8.0.

[0092] R2 reagent includes: phosphate buffer 20 mmol / L, glycerol 350 mL / L, block polyether F68 1.5 g / L, Surfynol 465 1.5 g / L, Proclin 300 1.5 g / L, ADPN mixed antibody (ADPN antibody I: ADPN antibody II = 1:1) 100 mg / L, 400 nm latex microsphere particles 1.5%, pH 8.0.

[0093] Calibrators: The calibrators include: PBS buffer 20 mmol / L, sodium chloride 10 g / L, BSA 15 g / L, sodium azide 1.5 g / L, ADPN antigen 0-40 mg / L, pH 8.0.

[0094] Preparation method:

[0095] (1) Prepare R1 reagent:

[0096] According to the component content of R1 reagent, each component is mixed in the same container, and after mixing evenly, R1 reagent is prepared.

[0097] (2) Preparation of latex particles coated with ADPN antibody:

[0098] ① Add the latex microspheres to the coupling buffer (20 mmol / L Hepes buffer, pH 7.5); then, preheat in a constant temperature water bath at 37°C for 20 min.

[0099] ② Add activator EDC solution (10 mg / mL) and NHS solution (50 mg / L), mix well, and place in a constant temperature stirrer to react for 30 minutes at 37°C.

[0100] ③ Add ADPN antibody I and ADPN antibody II, mix well, and place in a constant temperature stirrer to react for 60 minutes at 37°C.

[0101] ④ Add blocking solution 1 (sodium caseinate, 45 g / L) and blocking solution 2 (Tween 20, 100 mL / L), mix well, and place in a constant temperature stirrer to react for 24 h at 37°C.

[0102] ⑤ Centrifuge in a high-speed centrifuge at 20,000 rpm for 10 minutes at 4°C.

[0103] ⑥ Add an appropriate amount of preservation buffer (phosphate buffer 20mmol / L, glycerol 350mL / L, block polyether F681.5g / L, Surfynol 465 1.5g / L, Proclin300 1.5g / L), place in an ice water bath, and place in an ultrasonic cell disruptor to fully disperse for 10 minutes at a power of 200W.

[0104] (3) Prepare R2 reagent:

[0105] According to the component contents of the R2 reagent, the latex particles coated with the ADPN antibody and the remaining components are mixed in the same container and mixed evenly to prepare the R2 reagent.

[0106] (4) Preparation of calibration materials:

[0107] According to the component content of the calibrator, the components are mixed in the same container and mixed evenly to prepare 0-40 mg / L ADPN reagent calibrators, preferably ADPN reagent calibrators with concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L.

[0108] Example 6

[0109] A method for using the latex-enhanced immunoturbidimetric assay kit for determining adiponectin in this embodiment:

[0110] Pipette 3uL of sample, add 160uL of reagent R1, and incubate for 3 to 5 minutes; then add 40uL of reagent R2; after incubation for 20 seconds, use a fully automatic biochemical analyzer to measure the absorbance value A1 at a wavelength of 700nm; incubate for another 5 minutes, and detect the absorbance value A2 at the same wavelength (the reaction method is the endpoint method, the main wavelength is 700nm, the reaction direction is the forward reaction, the reaction temperature is 37°C, and the light path of the cuvette is 1.0cm); calculate the absorbance change value △A according to △A=A2-A1, and substitute △A into the linear relationship formula between the absorbance change value and the ADPN concentration to convert the ADPN content.

[0111] Among them, the linear relationship formula between the absorbance change value and the ADPN concentration is obtained by the following method:

[0112] Prepare calibrants with various concentration gradients.

[0113] Dilute the purified ADPN antigen with calibrator buffer to prepare calibrator concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L.

[0114] To each 3uL ADPN standard, 160uL of reagent R1 was added, mixed, and incubated for 3-5 minutes. Then, 40uL of R2 was added, mixed, and incubated for 20 seconds. The absorbance value A1 was measured at a wavelength of 700nm. After 5 minutes, the absorbance value A2 was measured at a wavelength of 700nm (the reaction method was endpoint, the dominant wavelength was 700nm, the reaction direction was positive, the reaction temperature was 37°C, and the cuvette light path was 1.0cm). The absorbance change value ΔA = A2 - A1 of each standard was calculated, and a calibration curve was plotted. The calibration curve was fitted using multi-point nonlinear fitting to derive a linear relationship between the absorbance change value and the ADPN concentration.

[0115] Test Example 1

[0116] This test example is used to verify the advantages of the antibody of the present invention in sensitivity and accuracy compared with existing antibodies.

[0117] Experimental and control groups were set up, and calibration measurements and sensitivity and accuracy tests were performed respectively. Among them, the experimental group was an adiponectin assay kit using the self-developed ADPN antibody pair of the present invention (ADPN antibodies I and II) (taking the corresponding kit of Example 4 as an example). The control group was an adiponectin assay kit using a purchased commercial ADPN antibody pair (Wuhan Huamei Bioengineering Co., Ltd., adiponectin ADPN monoclonal antibody 1 "CSB-DA120AmN①", adiponectin ADPN monoclonal antibody 3 "CSB-DA120AmN③") (except for the replacement of antibodies, the rest was the same as Example 4).

[0118] 1. Calibration measurement

[0119] The calibration results of the control group and the experimental group are shown in Table 1. The calibration curves are shown in Figure 1 shown.

[0120] Table 1 Calibration results (unit: mg / L)

[0121]

[0122] From Table 1 and Figure 1 It can be seen that the sensitivity of the experimental group of the present invention (with self-developed antibody pair) is higher.

[0123] 2. Sensitivity test

[0124] A 10 mg / L sample was taken as the test sample, and the absorbance value was measured under the conditions of a dominant wavelength of 700 nm and a light path of 1.0 cm. The measurement was repeated twice. The results are shown in Table 2.

[0125] Table 2 Sensitivity results (unit: mg / L)

[0126]

[0127] As shown in Table 2, the sensitivity of the experimental group of the present invention (with the self-developed antibody pair) is higher.

[0128] 3. Accuracy test

[0129] Two clinical samples of different concentrations were tested using the experimental and control group reagents, and the relative deviations between the test values ​​and the target values ​​of the serum samples were calculated. The results are shown in Table 3.

[0130] Table 3 Accuracy results (unit: mg / L)

[0131] Grouping Target value control group Relative deviation Experimental group Relative deviation Clinical sample S1 5mg / L 4.5 -10％ 5.1 2％ Clinical sample S2 10mg / L 12.3 23％ 10.1 1％

[0132] As shown in Table 3, the experimental group of the present invention (with self-developed antibody pair) has higher accuracy.

[0133] In summary, the antibody pair used in the present invention has higher sensitivity and accuracy than the reagent prepared in the control group (purchased commercial antibody pair).

[0134] Test Example 2

[0135] This test example is used to verify the effect of adding block polyether F68, Surfynol 465, and glycerol on the stability of the reagent.

[0136] Four experimental groups were set up, and the reagents in each group were stored at 4°C in the dark. The quality control product with a target value of 10 mg / L was tested during the period of 1 to 13 months (see Example 6 for the test method).

[0137] Experimental group 1: adiponectin assay kit of the present invention (taking the corresponding kit of Example 4 as an example);

[0138] Experimental Group 2: Adiponectin assay kit with the segmented polyether F68 removed (i.e., the segmented polyether F68 was removed from the kit of Example 4);

[0139] Experimental group 3: an adiponectin assay kit with Surfynol 465 removed (i.e., Surfynol 465 was removed from the kit of Example 4);

[0140] Experimental Group 4: Adiponectin assay kit with glycerol removed (ie, glycerol was removed from the kit of Example 4).

[0141] The test results are shown in Table 4.

[0142] Table 4 Test stability results (unit: mg / L)

[0143]

[0144]

[0145] The results showed that experimental group 2, which removed block polyether F68 from the reagent, showed a gradual downward trend in the control sample's test values ​​from January to 13 months, with a significant decrease, indicating poor reagent stability. Experimental group 3, which removed Surfynol 465 from the reagent, also showed a gradual downward trend in the control sample's test values ​​from January to 13 months, but the decrease was smaller than that of experimental group 2. Experimental group 4, which removed glycerol from the reagent, also showed a gradual downward trend in the control sample's test values ​​from January to 13 months, with the smallest decrease compared to experimental groups 2 and 3. Experimental group 1, which contained Surfynol 465, block polyether F68, and glycerol in the reagent, showed minimal fluctuation in the control sample's test values ​​from January to 13 months, with the fluctuation within 2%, indicating good reagent stability.

[0146] In summary, the pair of adiponectin antibodies designed in the present invention (ADPN antibody I and ADPN antibody II) can bind to ADPN in serum, and the binding can increase the activity of ADPN, thereby significantly improving the sensitivity, accuracy, stability and repeatability of the reagent in the ADPN latex immunoturbidimetric assay. At the same time, the present invention adds glycerol, block polyether F68 and Surfynol 465 as stabilizers to the kit to further improve the stability of the test.

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A pair of adiponectin antibodies, characterized in that It includes ADPN antibody I and ADPN antibody II; the ADPN antibody I includes a light chain and a heavy chain, and the corresponding amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the ADPN antibody II includes a light chain and a heavy chain, and the corresponding amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. The adiponectin antibody according to claim 1, characterized in that The ADPN antibody I and ADPN antibody II can bind to adiponectin in serum and increase the activity of adiponectin after binding.

3. A latex-enhanced immunoturbidimetric assay kit for determining adiponectin, characterized in that: The invention comprises the adiponectin antibody according to claim 1 or 2.

4. The latex-enhanced immunoturbidimetry kit for determining adiponectin according to claim 3, wherein Specifically including R1 reagent and R2 reagent; The R1 reagent includes: Tris buffer 10-100 mmol / L, sodium chloride 10-50 g / L, Proclin 300 0.5-1.5 g / L, Tween-20 1-5 g / L, PEG 6000 10-30 g / L, pH 7.0-8.0; The R2 reagent comprises: phosphate buffer 5-20 mmol / L, glycerol 250-350 mL / L, block polyether F68 0.5-1.5 g / L, Surfynol 465 0.5-1.5 g / L, Proclin 300 0.5-1.5 g / L, ADPN mixed antibody 30-100 mg / L, 150-400 nm latex microsphere particles 0.5%-1.5%, pH 7.0-8.0; Wherein, the ADPN mixed antibody is a mixed antibody of ADPN antibody I and ADPN antibody II.

5. The latex-enhanced immunoturbidimetry kit for determining adiponectin according to claim 4, wherein The R1 reagent specifically includes: Tris buffer 50 mmol / L, sodium chloride 25 g / L, Proclin 300 1 g / L, Tween-20 1.5 g / L, PEG 6000 20 g / L, pH 7.

4.

6. The latex-enhanced immunoturbidimetry kit for determining adiponectin according to claim 4, characterized in that: The R2 reagent specifically includes: phosphate buffer 10 mmol / L, glycerol 300 mL / L, block polyether F68 1 g / L, Surfynol 465 1 g / L, Proclin 300 1 g / L, ADPN mixed antibody 60 mg / L, 250 nm latex microsphere particles 0.5%, pH 7.

4.

7. The latex-enhanced immunoturbidimetric assay kit for adiponectin determination according to claim 4, characterized in that: In the ADPN mixed antibody, the mass ratio of ADPN antibody I to ADPN antibody II is 1:

1.

8. The latex-enhanced immunoturbidimetry kit for determining adiponectin according to claim 4, characterized in that: In the R2 reagent, the ADPN mixed antibody is coupled with the latex microsphere particles to form latex particles coated with the ADPN antibody.

9. The latex-enhanced immunoturbidimetry kit for determining adiponectin according to claim 8, characterized in that: The preparation method of the latex particles coated with ADPN antibody is as follows: ① Add the latex microspheres to the coupling buffer; then, preheat in a constant temperature water bath; ② Add activator EDC solution and NHS solution, mix well, and place in a constant temperature stirrer for reaction; ③ Add ADPN antibody I and ADPN antibody II, mix well, and place in a constant temperature stirrer for reaction; ④ Add blocking solution, mix well, and place in a constant temperature stirrer for reaction; ⑤ Place in centrifuge for centrifugation; ⑥Add preservation buffer, place in an ice water bath, place in an ultrasonic cell disruptor, and fully disperse.

10. The latex-enhanced immunoturbidimetric assay kit for adiponectin determination according to claim 4, characterized in that: It also includes calibrators; the calibrators include: PBS buffer 5-20 mmol / L, sodium chloride 5-10 g / L, BSA 5-15 g / L, sodium azide 0.5-1.5 g / L, ADPN antigen 0-40 mg / L, pH 7.0-8.0.

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