Adiponectin chemiluminescence immunoassay kit and detection method and application thereof

Abstract

The invention discloses an adiponectin chemiluminescence immunoassay kit, which comprises the following components: a magnetic particle coating substance coated with a mouse anti-adiponectin monoclonal antibody; an enzyme marker, wherein the enzyme marker comprises a mouse anti-adiponectin monoclonal antibody labeled by alkaline phosphatase; the luminous substrate liquid is used for reacting with the enzyme marker to generate an optical signal; during detection, a sample and a calibrator are added into the reaction cup; sequentially adding a magnetic particle coating substance and an enzyme marker, and uniformly mixing; cleaning after incubating for preset time; adding a luminous substrate solution, and uniformly mixing; detecting a chemiluminescence signal, and calculating the adiponectin concentration in the sample through the calibration curve; therefore, high-sensitivity chemiluminescence detection is realized, the reaction efficiency can be improved, and a target substance with extremely low concentration can be detected; meanwhile, by adopting the kit, one-step detection can be realized, and the detection efficiency is further improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and particularly to a chemiluminescent immunoassay kit for adiponectin, its detection method and application. Background Art

[0002] Adiponectin (ADPN) is a protein hormone secreted by adipocytes and plays an important role in regulating fat and glucose metabolism, inflammatory responses, and cardiovascular diseases.

[0003] In existing adiponectin detection technologies, most are enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, and latex-enhanced immunoturbidimetry. There are some deficiencies in the traditional adiponectin detection methods currently used in the market: on the one hand, low sensitivity: in traditional detection technologies, ELISA relies on enzymatic reactions and is easily affected by chromogenic substrates, resulting in low sensitivity and a narrow linear range of detection results. For immunoturbidimetry and latex-enhanced turbidimetry, since their principle is to detect the turbidity caused by antigen-antibody binding to detect the adiponectin concentration in the sample, they are easily affected by other substances in the sample and have low sensitivity. On the other hand, cumbersome operation: traditional detection technologies such as ELISA highly rely on manual operation and consume a long time; turbidimetry usually requires the use of semi-automatic instruments and still needs manual assistance to a certain extent, unable to achieve rapid detection, which limits its application in clinical rapid diagnosis to a certain extent.

[0004] Therefore, a detection method with advantages such as high sensitivity, good specificity, and high automation degree is needed to meet the requirements of clinical rapid diagnosis. Summary of the Invention

[0005] To solve the above problems, one of the objectives of the present invention is to provide a chemiluminescent immunoassay kit for adiponectin. This kit realizes highly sensitive chemiluminescent detection through the combination of an enzyme label and a direct coating form, can improve the reaction efficiency and detect extremely low concentrations of target substances; at the same time, using the kit of the present invention can achieve one-step detection, further improving the detection efficiency.

[0006] To achieve the above objective, the technical solution adopted by the present invention is as follows: A chemiluminescent immunoassay kit for adiponectin, comprising the following components: Magnetic particle coating, coated with mouse anti-adiponectin monoclonal antibody; Enzyme label, the enzyme label includes mouse anti-adiponectin monoclonal antibody labeled with alkaline phosphatase; Luminescence substrate solution, used to react with the enzyme label to generate a light signal.

[0007] Preferably, it further includes: Calibrator series: Using a calibrator buffer solution to prepare a series of standard solutions with different concentrations of adiponectin antigen; Quality control product: The matrix solution uses MES buffer, and adiponectin is dissolved in the MES buffer to prepare a quality control product solution with a target concentration.

[0008] Preferably, the preparation of the calibrator includes the following steps: Prepare the calibrator buffer: Use a buffer containing BSA, MES-Na, MES, ProClin 300, and purified water as the matrix; Prepare the adiponectin standard solution: Dissolve natural adiponectin in the calibrator buffer to prepare a series of standard solutions with different concentrations, including 0 ng / mL, 5 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, and 2000 ng / mL.

[0009] Subpackage and preservation: Subpackage the prepared standard solution into small tubes, freeze-dry after subpackaging a fixed volume per tube, and store for later use.

[0010] Preferably, it further includes a sample diluent, and the sample diluent is composed of the following components: 0.7 - 0.8% sodium chloride, 20% newborn bovine serum, 0.1 - 0.3% Proclin-300, 0.4 - 0.6% Tris, 0.2 - 0.3% HCl, and purified water.

[0011] Preferably, it further includes a concentrated cleaning solution, and the concentrated cleaning solution is composed of the following components: 18% - 20% NaCl, 2.4 - 3.0% Tris, 0.4 - 0.6% MgCl2·6H2O, 0.95 - 1% HCl, and a preservative; among them, the preservative uses 0.5% Krovin600 or 1% NaN3, and 2 - 3% Triton X-100.

[0012] Preferably, the chemiluminescent substrate solution is composed of the following components: a chemiluminescent substrate for alkaline phosphatase - CSPD, a buffer system containing Tris, a preservative, and purified water.

[0013] Preferably, the preparation of the magnetic particles includes the following steps: Magnetic particle pretreatment: After mixing the magnetic particles, remove the supernatant by magnetic separation; Washing and dilution: Wash the magnetic particles with a buffer solution more than once, and then dilute the magnetic particles to the target concentration; Antibody coating: Add an antibody to the magnetic particle suspension for reaction to achieve antibody coating; Washing and blocking treatment: Wash the coated magnetic particles with a buffer salt solution more than once to remove unbound components; add a buffer solution containing a blocking agent at a first concentration for reaction to block the unbound sites on the surface of the magnetic particles; Washing and storage: Wash the magnetic particles with a buffer salt solution again more than once to remove the excess blocking agent; add a buffer solution containing a blocking agent at a second concentration for storage; wherein, the second concentration is less than the first concentration.

[0014] Preferably, the preparation of the enzyme label includes the following steps: Activation of the enzyme: Purify alkaline phosphatase, chemically modify alkaline phosphatase with an activation reagent, and then remove the excess activation reagent; Activation of the antibody: Purify the adiponectin monoclonal antibody, chemically modify the adiponectin monoclonal antibody with an activation reagent, and then remove the excess activation reagent; Labeling reaction: Mix the activated alkaline phosphatase with the activated adiponectin monoclonal antibody for a labeling reaction. After the reaction is completed, add a blocking agent for blocking treatment; Purification and storage: Purify the labeled product, add a protective agent, and store for later use.

[0015] The second object of the present invention is to provide a detection method for the adiponectin chemiluminescence immunoassay kit described in any one of the above, which includes the following steps: Add the sample and the calibrator to the reaction cup and dilute; Add the magnetic particle coating and the enzyme label in sequence and mix well; Wash after incubation for a preset time; Add the luminescent substrate solution and mix well; Detect the chemiluminescence signal and calculate the concentration of adiponectin in the sample through the calibration curve.

[0016] The third object of the present invention is to provide the application of the adiponectin chemiluminescence immunoassay kit described in any one of the above in a chemiluminescence analyzer.

[0017] The beneficial effects of the present invention specifically include: (1), In the kit of the present invention, alkaline phosphatase is used as the enzyme label to label the mouse anti-adiponectin monoclonal antibody. Its efficiency of catalyzing the substrate to generate chemiluminescence signal is high, which can significantly improve the detection sensitivity, so that the presence of adiponectin can be detected even at low concentrations; moreover, the catalytic reaction rate of alkaline phosphatase is fast, and sufficient light signals can be generated in a short time, shortening the detection time and improving the detection efficiency.

[0018] (2) The present invention uses natural adiponectin as a standard, which is closer to adiponectin in actual samples, improving the accuracy and relevance of calibration. By preparing a series of standard solutions with different concentrations covering a range from low to high concentrations, different detection requirements are met, ensuring calibration of the detection system within a wide linear range and improving the applicability of the detection system.

[0019] (3) The sample diluent of the present invention ensures the stability of adiponectin in the sample during dilution by optimizing its components, reducing degradation or denaturation. Even at low concentrations, adiponectin can maintain its biological activity, ensuring the accuracy of the detection results. Among them, newborn bovine serum and Tris buffer can provide a stable protein environment, reducing non-specific binding of adiponectin in the sample to other components of the kit, reducing background signals, and improving the specificity and sensitivity of the detection.

[0020] (4) The sample diluent can not only effectively protect adiponectin in the sample and improve the sensitivity of the detection, but also enable detection with a smaller sample volume, reducing the sample requirement, lowering the detection cost, and avoiding detection failure due to insufficient samples.

[0021] (5) The concentrated washing solution, through high concentrations of NaCl and Tris buffer, can effectively remove unbound components, reduce background signals, and improve the specificity and sensitivity of the detection. The addition of MgCl2 optimizes the chemical environment of the detection system, ensuring the activity of the enzyme and the stability of the protein, thereby improving the reliability of the detection results.

[0022] (6) In the luminescent substrate solution, CSPD can generate a strong chemiluminescent signal under the catalysis of alkaline phosphatase, enabling the detection of the presence of the target substance even at low concentrations, significantly improving the sensitivity of the detection, and being able to detect adiponectin concentrations as low as 0.15 ng / mL.

[0023] (7) The preparation process of the magnetic microparticles ensures the high specificity, stability, and sensitivity of the magnetic microparticles through multiple steps of operation such as pretreatment, washing, antibody coating, blocking treatment, and preservation. Among them, through antibody coating and blocking treatment, the magnetic microparticles can specifically bind to adiponectin, reducing non-specific binding and improving the sensitivity and specificity of the detection; washing and blocking treatment ensure the cleanliness and stability of the surface of the magnetic microparticles, reducing background signals caused by impurities or unbound sites; using a blocking agent to preserve the magnetic microparticles extends their shelf life and reduces antibody shedding or denaturation due to improper storage.

[0024] (8) The preparation process of the enzyme label ensures high specificity, stability, and sensitivity of the label through steps such as enzyme activation, antibody activation, labeling reaction, purification, and preservation. Among them, the alkaline phosphatase is purified, chemically modified with an activation reagent (such as SMCC), and then the excess activation reagent is removed, which can ensure that the alkaline phosphatase has high activity and reactivity, providing a basis for the subsequent labeling reaction; the adiponectin monoclonal antibody is purified, chemically modified with an activation reagent, enabling the antibody to covalently bind to the enzyme, ensuring that the antibody has high reactivity and can efficiently bind to the alkaline phosphatase. Detailed implementation manners

[0025] In order to make the technical problems, technical solutions, and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] In addition, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. The intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention.

[0028] The source information of some reagents in the embodiments of the present invention is shown in Table 1.

[0029] Table 1 Main reagents

[0030] First embodiment (kit): This embodiment provides an adiponectin chemiluminescent immunoassay kit, which includes the following components: Magnetic particle coating, coated with mouse anti - adiponectin monoclonal antibody; Enzyme label, the enzyme label includes alkaline phosphatase - labeled mouse anti - adiponectin monoclonal antibody; Luminescence substrate solution, used to react with the enzyme label to generate a light signal.

[0031] Calibration standard series: A series of standard solutions with different concentrations of adiponectin antigen were prepared using calibration standard buffer; the concentrations of the standard solutions include 0 ng / mL, 5 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, and 2000 ng / mL.

[0032] Quality control sample: The matrix solution is 50 mmol / L MES buffer, and adiponectin is dissolved in the MES buffer to prepare a quality control sample solution with the target concentration.

[0033] Sample diluent (optional): The sample diluent consists of the following components: 0.7 - 0.8% sodium chloride, 20% newborn bovine serum, 0.1 - 0.3% Proclin-300, 0.4 - 0.6% Tris, 0.2 - 0.3% HCl, and purified water. By mixing serum and plasma samples with the buffer, the concentrations of certain chemicals in the serum and plasma are reduced to be within the limited range of detection.

[0034] Concentrated washing solution (optional); The concentrated washing solution consists of the following components: 18% - 20% NaCl, 2.4 - 3.0% Tris, 0.4 - 0.6% MgCl₂·6H₂O, 0.95 - 1% HCl (36 - 38%), and a preservative; among them, the preservative is 0.5% Krovin600 or 1% NaN₃, 2 - 3% Triton X-100.

[0035] The chemiluminescent substrate solution consists of the following components: a chemiluminescent substrate for alkaline phosphatase - CSPD, a buffer system containing Tris, a preservative, and purified water.

[0036] Alkaline Phosphatase (AP) is an enzyme widely present in various biological tissues. Its main function is to catalyze the hydrolysis of phosphate ester bonds, removing the phosphate group from the substrate to generate the corresponding alcohol or phenolic product. In the present invention, alkaline phosphatase is used as a labeled enzyme and covalently binds to the adiponectin monoclonal antibody, which can catalyze the chemiluminescent substrate to produce a light signal, significantly improving the detection sensitivity; by binding to the antibody, alkaline phosphatase can specifically recognize adiponectin, reducing non-specific binding; the catalytic reaction of alkaline phosphatase is fast, and it can generate sufficient signals in a short time, shortening the detection time.

[0037] Mouse anti-adiponectin monoclonal antibody, namely Mouse Anti-Adiponectin Monoclonal Antibody, is a monoclonal antibody that specifically recognizes adiponectin and is prepared by the hybridoma technology, which can bind to adiponectin molecules with high specificity.

[0038] Mouse anti - adiponectin monoclonal antibody is a monoclonal antibody that specifically recognizes adiponectin, prepared by immunizing mice and screening hybridoma cells: Mice are immunized by multiple injections of adiponectin antigen to induce an immune response and produce specific antibodies. The antigen is usually recombinant adiponectin protein or adiponectin peptide fragments; B cells are extracted from the mouse spleen and fused with myeloma cells (such as SP2 / 0) to form hybridoma cells. After fusion, hybridoma cell clones that can secrete specific antibodies are screened; Hybridoma cell clones with high specificity and high affinity are screened by methods such as ELISA, and the antibody is extracted from cell culture supernatant or mouse ascites and purified. Mouse anti - adiponectin monoclonal antibody has high specificity, can specifically recognize adiponectin molecules, and has no obvious cross - reaction with other proteins or adipokines; Moreover, monoclonal antibodies have high affinity, can efficiently bind adiponectin, and can detect the target protein even at low concentrations.

[0039] Tris - HCl buffer (Tris - Hydrochloric Acid Buffer) is composed of tris (hydroxymethyl) aminomethane (Tris, also known as tris (hydroxymethyl) aminomethane or 2 - amino - 2 - (hydroxymethyl) - 1,3 - propanediol) and hydrochloric acid (HCl), and is used to maintain the pH value of the solution stable, ensuring that biomolecules remain stable and active under suitable pH conditions. By providing a stable pH environment, Tris - HCl buffer reduces non - specific binding and improves the specificity of detection. The effective pH range of Tris - HCl buffer is usually between 7.0 and 9.0, and the specific pH value is adjusted by adding appropriate amounts of HCl or NaOH.

[0040] PBS buffer (Phosphate Buffered Saline) is also used to maintain the pH value of the solution stable, ensuring that biomolecules (such as proteins, enzymes, etc.) remain stable and active under suitable pH conditions. PBS buffer: It is composed of disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl) and water, and is used to maintain the pH value of the solution in the neutral range (usually pH 7.2 to 7.6).

[0041] MES buffer (2 - (N - morpholino) ethanesulfonic acid buffer, Morpholine Ethanesulfonic Acid Buffer) is a buffer solution prepared from 2 - (N - morpholino) ethanesulfonic acid (MES) and appropriate amounts of acid or base, and is used to maintain the pH value of the solution in the acidic range (usually pH 5.5 to 7.0).

[0042] Second Embodiment (Preparation Methods of Each Component): (1). The preparation of magnetic particles includes the following steps: Magnetic particle pretreatment: After mixing the magnetic particles evenly, remove the supernatant by magnetic separation; Washing and dilution: Wash the magnetic particles with a buffer solution more than once, and then dilute the magnetic particles to the target concentration; Antibody coating: Add an antibody to the magnetic particle suspension for reaction to achieve antibody coating; Washing and blocking treatment: Wash the coated magnetic particles with a buffer salt solution more than once to remove unbound components; Add a buffer solution containing a blocking agent at the first concentration for reaction to block the unbound sites on the surface of the magnetic particles; Washing and storage: Wash the magnetic particles with a buffer salt solution again more than once to remove the excess blocking agent; Add a buffer solution containing a blocking agent at the second concentration for storage; wherein, the second concentration is less than the first concentration.

[0043] Specifically, it includes the following steps: a1. Pretreatment of magnetic particles: Place the tosylated magnetic particles with a particle size of 1.0 - 1.2 μm on a rotary mixer, set the rotation speed to 40 - 60 rpm, and mix evenly for more than half an hour; Transfer 10 mg - 25 mg of magnetic particles to a reagent bottle, place the reagent bottle on a magnetic separator, and after the magnetic particles are completely adsorbed, remove the supernatant; a2. Washing and dilution of magnetic particles: Add a borate buffer solution with a pH of 9.0 - 10.0, wash the magnetic particles repeatedly 3 times, and remove the supernatant after each wash; Add a borate buffer solution to dilute the magnetic particles to 10 mg / mL; a3. Antibody coating: Add adiponectin antibody to the magnetic particle suspension at a coating concentration of 10 - 25 μg / mg, then add 0.5 - 1 mL of 3M ammonium sulfate solution, and rotate and mix evenly at 37 ± 2 °C for 16 - 24 hours, and remove the supernatant; a4. Blocking treatment: Add a TBST buffer salt solution with a pH of 7.0 - 8.0, wash the magnetic particles repeatedly 3 times, and remove the supernatant after each wash; Add a TBST buffer salt solution containing 1% (the first concentration) bovine serum albumin (BSA), rotate and mix evenly at 37 ± 2 °C for 16 - 24 hours, and remove the supernatant; a5. Storage: Add a TBST buffer salt solution with a pH of 7.0 - 8.0, wash the magnetic particles repeatedly 3 times, and remove the supernatant after each wash; Add a TBST buffer salt solution containing 0.1% (the second concentration) bovine serum albumin (BSA) to obtain magnetic particles coated with mouse anti - adiponectin monoclonal antibody at 10 mg / mL, and store them for standby at 2 - 8 °C.

[0044] Among them, tosylation is a chemical modification process used for functionalizing compounds or biomolecules. The tosyl group (with the chemical formula CH3C6H4SO3) is a protecting group or activating group that can introduce specific chemical properties or reaction sites. In this example, the tosyl group is introduced onto the surface of magnetic microparticles to facilitate subsequent antibody coating. The tosyl group can act as an activating group and undergo a coupling reaction with the amino group of the antibody to form a stable covalent bond. The introduction of the tosyl group can increase the stability and reactivity of magnetic microparticles, and the tosylated magnetic microparticles can efficiently bind antibodies, improving the antibody coating efficiency.

[0045] The 3M ammonium sulfate solution has high solubility and good buffering capacity. It is weakly acidic in aqueous solution and can provide a stable ionic environment. During the process of coating antibodies on the surface of magnetic microparticles, the ammonium sulfate solution neutralizes the negative charges on the antibodies, reduces electrostatic repulsion, makes it easier for the antibodies to adsorb onto the surface of magnetic microparticles, significantly improves the antibody coating efficiency, and enhances the binding stability between the antibodies and magnetic microparticles.

[0046] (2). The preparation of the enzyme-labeled product includes the following steps: Activation of the enzyme: Purify alkaline phosphatase, chemically modify alkaline phosphatase using an activating reagent, and then remove the excess activating reagent; Activation of the antibody: Purify the adiponectin monoclonal antibody, chemically modify the adiponectin monoclonal antibody using an activating reagent, and then remove the excess activating reagent; Labeling reaction: Mix the activated alkaline phosphatase with the activated adiponectin monoclonal antibody for the labeling reaction. After the reaction is completed, add a blocking agent for blocking treatment; Purification and preservation: Purify the labeled product, add a protective agent, and store it for later use.

[0047] Specifically, it includes the following steps: b1. Dialyze alkaline phosphatase in a buffer solution with a pH of 7.2 - 8 for 6 - 24 h (or use a desalting column or ultrafiltration). Prepare a solution of N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) before use, with DMSO as the solvent. Add SMCC to alkaline phosphatase, shake and react at room temperature for 0.5 - 2 h. Dialyze the reacted alkaline phosphatase in a buffer solution with a pH of 7.2 - 7.5 for 6 - 24 h (or use a desalting column or ultrafiltration) to remove the excess SMCC; b2. Dialyze the antibody or antigen in a buffer solution with a pH of 7 - 9.5 and containing 1 - 5 mM EDTA for 6 - 24 h (or use a desalting column or ultrafiltration). Prepare a 2-iminothiolane hydrochloride solution (2-IT·HCl) before use. Add 2-IT·HCl to the mouse adiponectin monoclonal antibody at a concentration of 1 - 2 mg / mL, and react with shaking at room temperature for 1 - 2 h. After the reaction, dialyze (using a desalting column or ultrafiltration) in a buffer solution with a pH of 7.2 - 7.5 to remove the excess 2-IT·HCl; b3. After activation, mix the SMCC-activated alkaline phosphatase with the 2-IT·HCl-activated adiponectin antibody, and react with shaking at room temperature for 1 - 4 h; after the reaction, add bovine serum albumin (BSA) to a concentration of 0.1 mg / mL; b4. Dialyze (or desalt, ultrafilter) in 0.05 M Tris-HCl (pH 7.0 - 8), add an equal volume of glycerol to the alkaline phosphatase-labeled adiponectin antibody, and store at -20 °C for later use.

[0048] Among them, 2-iminothiolane hydrochloride (2-Iminothiolane·HCl, abbreviated as 2-IT·HCl) is a chemical modification reagent mainly used to introduce reactive groups such as sulfhydryl (-SH) or amino (-NH2) to modify the mouse adiponectin monoclonal antibody, enabling it to react with the activated groups (such as tosyl groups) on the surface of magnetic particles to achieve efficient coating.

[0049] The EDTA (Ethylene Diamine Tetraacetic Acid) buffer solution is mainly used to adjust the metal ion concentration in the solution or inhibit the activity of metal ion-dependent enzymes. By adjusting the reaction conditions and preventing metal ion interference, the specificity and sensitivity of the detection are ensured.

[0050] (3). The preparation of the calibrator includes the following steps: Prepare the calibrator buffer solution: Use a buffer solution containing BSA, MES-Na, MES, ProClin 300, and purified water as the matrix; Prepare the adiponectin standard solution: Dissolve natural adiponectin in the calibrator buffer solution to prepare a series of standard solutions with different concentrations, including 0 ng / mL, 5 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, and 2000 ng / mL.

[0051] Aliquot and storage: Aliquot the prepared standard solution into small tubes, freeze-dry after aliquoting a fixed volume for each tube, and store for later use; preferably, aliquot 0.5 mL into each tube, and after freeze-drying, store at 2 °C - 8 °C for later use.

[0052] BSA (Bovine Serum Albumin): As a stabilizer, it prevents adiponectin from aggregating or degrading in the buffer, ensuring the stability of the standard solution.

[0053] MES-Na and MES: Provide a suitable buffering environment, maintain the acid-base balance of the solution, and ensure the solubility and stability of adiponectin.

[0054] ProClin 300: As a preservative, it prevents microbial contamination and extends the shelf life of the calibrator.

[0055] Purified water: As a solvent, it provides a clean matrix and avoids interference from impurities in the test results.

[0056] Among them, natural adiponectin is extracted and purified from adipose tissue (such as mouse or human fat). The adipose tissue is digested with collagenase to separate the stromal vascular fraction, and after non-denaturing lysis, it is gradually purified by ammonium sulfate precipitation, ion exchange chromatography (such as DEAE column), heparin affinity chromatography, and gel filtration chromatography (such as Superdex 200) in sequence to retain its natural multimeric structure; the whole purification process needs to be carried out at low temperature and protease inhibitors are added to avoid the destruction of the conformation by strong denaturants. The purified adiponectin needs to be verified for purity and specificity by SDS-PAGE and Western Blot, and concentrated by ultrafiltration. After aliquoting, it is stored at -80 °C (5% glycerol or 1% BSA can be added to prevent aggregation) to ensure its biological activity and stability.

[0057] The third embodiment (detection method): The present invention also provides a detection method for the above-mentioned adiponectin chemiluminescent immunoassay kit, which includes the following steps: Add the sample and the calibrator to the reaction cup and dilute; Add the magnetic particle conjugate and the enzyme conjugate in sequence and mix well; Incubate for a preset time and then wash; Add the luminescent substrate solution and mix well; Detect the chemiluminescent signal and calculate the concentration of adiponectin in the sample through the calibration curve.

[0058] The specific test process is as follows: 1). Place the sample container in the sample rack provided with the instrument, and keep the sample volume for each test above 200 μL; 2). Load the sample rack and input the sample information in the instrument software interface; 3). Select "Run" to start the test, and the system will automatically perform the test operation; 4). The sample rack is transferred to the sampling position; 5). Load the reaction cup to the sample addition position; 6). Add the calibrator and the sample into the reaction cup in sequence, with the sample addition volume being 8 μL / cup (30-fold dilution). 7). Add 50 μL of the magnetic particle suspension into each cup respectively. 8). Add 50 μL of the enzyme label into each cup respectively. 9). Incubate for 5 minutes after mixing. 10). Wash with the washing solution. 11). Add the substrate solution for the immunoassay system into each cup, mix well, and detect the chemiluminescence signal. 12). Result calculation: The instrument automatic operating system can automatically calculate the sample test result through the stored calibration curve and the luminescence intensity value obtained from the sample test; the result unit is given in the form of μg / mL.

[0059] Among them, the sample requirements are as follows: 1). The sample types detected by this reagent are human serum, heparinized plasma, and EDTA-anticoagulated plasma. 2). When the sample contains 10 g / L hemoglobin, 20 mg / mL fat emulsion, or 342 μmol / L bilirubin, it has no effect on the test result, but it is still recommended not to use samples with severe hemolysis, lipemia, or jaundice. 3). The sample should be centrifuged thoroughly and should not have hemolysis and cell particles. 4). The sample can be stored for 7 days at 2 - 8 °C and for 12 months at -20 °C and below. Avoid repeated freezing and thawing.

[0060] The performance indicators of the detection method of the present invention are specifically as follows: 1). Accuracy: The recovery rate is within the range of 85% - 115%. 2). Linearity: In the concentration range of 5 ng / mL to 2000 ng / mL, the correlation coefficient r should not be less than 0.9900.

[0061] Implementation method: Repeat the detection of each concentration of the enterprise linear reference product 2 times, calculate the average value, perform linear fitting on the result average value and the theoretical value by the least squares method, and calculate the linear correlation coefficient r.

[0062] 3). Blank limit: Not greater than 0.15 ng / mL.

[0063] Implementation method: Parallelly determine the zero-value calibrator or sample diluent 20 times, record their signal values, calculate the mean value () and the standard deviation (s), and calculate the value of (+2s). The sample concentration corresponding to this value is the blank limit of this kit.

[0064] 4). Repeatability: Three precision reference standards with concentration levels of 10 μg / mL, 15 μg / mL, and 30 μg / mL were used for determination, and the within-batch precision of the determination results was <10%.

[0065] 5). Between-batch difference: Three precision reference standards with concentration levels of 10 μg / mL, 15 μg / mL, and 30 μg / mL were used for determination, and the between-batch precision was <15%.

[0066] 6). Homogeneity of calibrator: The coefficient of variation (CV) within and between bottles of the calibrator should not be higher than 8.0%.

[0067] 7). Homogeneity of quality control material: The coefficient of variation (CV) within and between bottles of the quality control material should not be higher than 8.0%.

[0068] Fourth Embodiment (Application): The present invention also provides the application of the above adiponectin chemiluminescent immunoassay kit in a chemiluminescent analyzer. In the chemiluminescent analyzer, after the sample is mixed with the reagent, a chemiluminescent reaction occurs, and the generated light signal is detected by a photomultiplier tube or a photodiode, and data processing and result analysis are performed through a software system.

[0069] The application of the adiponectin chemiluminescent immunoassay kit of the present invention in a chemiluminescent analyzer significantly improves the sensitivity, specificity, and efficiency of detection.

[0070] The present invention uses the chemiluminescent method and the double antibody sandwich method. The calibrator, sample, magnetic particles coated with mouse anti-adiponectin monoclonal antibody, and adiponectin antibody-alkaline phosphatase conjugate are added to the reaction cup. After reacting for a sufficient time, a mouse anti-adiponectin monoclonal antibody~adiponectin~mouse anti-adiponectin monoclonal antibody-alkaline phosphatase conjugate complex is formed, and the free components are washed away. The immunological detection system adds the substrate solution to the reaction cup, and alkaline phosphatase catalyzes the dephosphorylation of the luminescent substrate to generate chemiluminescence, and the luminescence value (RLU) of each sample is detected. The adiponectin content in the sample is proportional to the detected luminescence value (RLU). The adiponectin concentration of the sample is determined by the calibration curve. Using the kit of the present invention, it has high sensitivity and a wide linear range, reaching more than 2000 ng / mL; the sample volume of the sample to be tested is small, the consumption of raw materials is small, and the sample addition mode is 8 μL sample + 50 μL magnetic particle coating + 50 μL conjugate; compared with the prior art, the kit of the present invention adopts a direct coating form and the chemiluminescent label is alkaline phosphatase, the reaction time is about 5 min, the method is a one-step method, the operation is simple, the result is obtained quickly, the time consumption is small, and it is detected by a fully automatic chemiluminescent instrument, and the manual processing steps are few, achieving the effect of rapid detection, and the total time is about 15 min.

[0071] The foregoing description illustrates and describes preferred embodiments of the present invention. As previously mentioned, it should be understood that the present invention is not limited to the forms disclosed herein, should not be construed as excluding other embodiments, but can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the above teachings or the skills or knowledge in the relevant field. Any alterations and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A chemiluminescent immunoassay kit for adiponectin, characterized in that: The kit comprises the following components: Magnetic microparticles coated with mouse anti-adiponectin monoclonal antibody; An enzyme marker, wherein the enzyme marker comprises an alkaline phosphatase-labeled mouse anti-adiponectin monoclonal antibody; The luminescent substrate liquid is used to react with the enzyme marker to generate a light signal.

2. The adiponectin chemiluminescent immunoassay kit according to claim 1, characterized in that: Also includes: Calibrator series: Adiponectin antigen was prepared into a series of standard solutions with different concentrations using calibrator buffer; Quality control product: The matrix solution uses MES buffer. Adiponectin is dissolved in MES buffer to prepare a quality control solution of the target concentration.

3. The adiponectin chemiluminescent immunoassay kit according to claim 2, characterized in that: The preparation of the calibrator comprises the following steps: Prepare calibrator buffer: Use a buffer containing BSA, MES-Na, MES, ProClin 300, and purified water as the matrix; Preparation of adiponectin standard solution: Dissolve natural adiponectin in calibration buffer to prepare a series of standard solutions with different concentrations, including 0 ng / mL, 5 ng / mL, 20 ng / mL, 100 ng / mL, 500 ng / mL, and 2000 ng / mL. Packaging and storage: Pack the prepared standard solution into small tubes. After each tube is filled with a fixed volume, freeze-dry it and store it for future use.

4. The adiponectin chemiluminescent immunoassay kit according to claim 1, characterized in that: The invention also includes a sample diluent, which is composed of the following components: 0.7-0.8% sodium chloride, 20% newborn calf serum, 0.1-0.3% Proclin-300, 0.4-0.6% Tris, 0.2-0.3% HCl, and purified water.

5. The adiponectin chemiluminescent immunoassay kit according to claim 1, characterized in that: The invention also includes a concentrated cleaning solution, which is composed of the following components: 18%~20NaCl, 2.4~3.0%Tris, 0.4~0.6%MgCl2·6H2O, 0.95~1%HCL, and a preservative; wherein the preservative is 0.5%Krovin600 or 1%NaN3, and 2~3%Triton X-100.

6. The adiponectin chemiluminescent immunoassay kit according to claim 1, characterized in that: The luminescent substrate solution is composed of the following components: chemiluminescent substrate of alkaline phosphatase-CSPD, a buffer system containing Tris, a preservative, and purified water.

7. The adiponectin chemiluminescent immunoassay kit according to claim 1, characterized in that: The preparation of the magnetic particles comprises the following steps: Magnetic particle pretreatment: After mixing the magnetic particles, remove the supernatant by magnetic separation; Washing and dilution: Wash the magnetic particles one or more times with a buffer solution, and then dilute the magnetic particles to the target concentration; Antibody coating: adding antibodies to the magnetic particle suspension to react to achieve antibody coating; Washing and blocking treatment: washing the coated magnetic particles with a buffered saline solution for more than one time to remove unbound components; adding a buffer solution containing a first concentration of a blocking agent to react to block unbound sites on the surface of the magnetic particles; Washing and preservation: Wash the magnetic particles again with the buffered saline solution for more than one time to remove excess blocking agent; add a buffer solution with a second concentration of blocking agent and preserve for later use; wherein the second concentration is less than the first concentration.

8. The adiponectin chemiluminescent immunoassay kit according to claim 1, characterized in that: The preparation of the enzyme marker comprises the following steps: Enzyme activation: purification of alkaline phosphatase, chemical modification of alkaline phosphatase with an activation reagent, and subsequent removal of excess activation reagent; Activation of the antibody: purifying the adiponectin monoclonal antibody, chemically modifying the adiponectin monoclonal antibody with an activation reagent, and then removing excess activation reagent; Labeling reaction: Mix activated alkaline phosphatase and activated adiponectin monoclonal antibody to carry out labeling reaction. After the reaction is completed, add blocking agent for blocking treatment; Purification and preservation: The labeled product is purified, and after adding a protective agent, it is stored for future use.

9. The detection method of the adiponectin chemiluminescent immunoassay kit according to any one of claims 1 to 8, characterized in that: The following steps are involved: Add samples and calibrators to the reaction cup and dilute; Add magnetic particle coating and enzyme marker in sequence and mix well; Wash after incubation for a preset time; Add luminescent substrate solution and mix well; The chemiluminescent signal was detected and the adiponectin concentration in the sample was calculated using the calibration curve.

10. Use of the adiponectin chemiluminescent immunoassay kit according to any one of claims 1 to 8 in a chemiluminescent analyzer.

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