CN111239421A - Latex-enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN and preparation and application methods thereof - Google Patents

Latex-enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN and preparation and application methods thereof Download PDF

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CN111239421A
CN111239421A CN202010101938.8A CN202010101938A CN111239421A CN 111239421 A CN111239421 A CN 111239421A CN 202010101938 A CN202010101938 A CN 202010101938A CN 111239421 A CN111239421 A CN 111239421A
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adiponectin
latex particles
antibody
buffer
streptavidin
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芮双印
符修乐
邵家祥
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Anhui Daqian Bio Engineering Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Abstract

The invention provides a latex-enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN, which comprises an adiponectin R1 reagent and an adiponectin R2 reagent; the adiponectin R1 reagent comprises a buffer solution A, a protective agent A, a reaction enhancer and a preservative; the adiponectin R2 reagent comprises a buffer solution B, a protective agent B, a preservative and sensitized polystyrene latex particles coated with an anti-human adiponectin antibody; wherein the anti-human adiponectin antibodies in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibodies are connected with the sensitized polystyrene latex particles through a streptavidin-biotin system. The invention also provides a preparation method and an application method of the latex enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN. The invention has the advantages that: the kit is convenient and rapid, has high sensitivity and specificity, and can quantitatively and accurately detect ADPN; the device has strong instrument compatibility and low detection cost, and makes up the clinical requirement on ADPN turbidimetric products.

Description

Latex-enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN and preparation and application methods thereof
Technical Field
The invention relates to the technical field of biological detection, in particular to a latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN and a preparation and application method thereof.
Background
Adiponectin (Adiponectin/ADPN) is an endogenous, biologically active polypeptide or protein secreted by adipocytes. Adiponectin is An Insulin-sensitizing Hormone (An Insulin-sensitizing Hormone) and can improve Insulin resistance (Insulin resistance) and arteriosclerosis in mice; the research on human bodies finds that the adiponectin level can indicate the development of II type diabetes and coronary heart disease, and shows the potential of resisting diabetes, atherosclerosis and inflammation in clinical tests.
Researchers have discovered a new compound that regulates adiponectin and thus have proposed a new approach to study adiponectin function and insulin sensitivity mechanism, insulin is a hormone secreted from pancreatic β cells and has a primary function of promoting glucose in blood to enter muscle or adipose tissue, providing energy required by the human body, when insulin fails to function, glucose in blood cannot be converted to energy required by the human body, leading to elevated blood glucose, and diabetes thus occurs, while insulin resistance means that cells cannot effectively utilize insulin even are no longer sensitive to insulin, which is the leading cause of diabetes.
The fat factor is a cytokine and hormone with biological activity secreted by adipose tissue, and is closely related to metabolic processes such as insulin resistance to coronary atherosclerosis and the like. Wherein the adiponectin is the highest fat factor in peripheral blood, and the plasma adiponectin level in healthy people is 5-30 mug/mL. Adiponectin binds to its receptor, and then activates adenosine activated protein kinase (AMPK), p38 mitogen activated protein kinase, peroxisome proliferator-activated receptor (PPAR), and other signal molecules, thereby exerting various biological effects such as anti-inflammatory, anti-diabetic, and anti-atherosclerosis. In animal experiments, adiponectin knockout mice are more likely to have serious immunological rejection than normal mice, and the rejection is obviously improved after adiponectin injection, which indicates that adiponectin can reduce the immunological rejection of mice. While Wilk et al found that adiponectin had an inhibitory effect on antigen-activated effector T cells, it is presumed that adiponectin is involved in immune regulation. Rheumatic diseases generally refer to a group of diseases affecting bones, joints and surrounding soft tissues, the main pathological changes being inflammatory and non-inflammatory pathologies. Inflammatory reaction most of the rest of the inflammatory reaction is caused by immune reaction except that gouty arthritis is caused by uric acid crystallization. Further studies have shown that adiponectin is expressed in elevated levels in the plasma and synovial fluid of patients with rheumatoid arthritis, and that adiponectin levels in synovial fluid are inversely correlated with leukocyte counts. The expression level in the serum of the systemic lupus erythematosus patient is higher than that of a normal control patient, and is obviously related to the disease activity of the patient.
Adiponectin, among the biologically active class of protein factors secreted by adipocytes, is one of the most abundantly expressed protein products of adipose tissue genes, present in large amounts in the blood circulation, and is present in circulating plasma at a concentration of 3-30ug/ml in humans, adiponectin, also known as Acrp30, apM1, AdipoQ, GBP28, and initially, adiponectin, found in human subcutaneous adipose tissue, plasma and murine adipocytes, adiponectin, in humans, consists of 244 amino acids, 30 KD. of molecular weight, an amino-terminal secretory signal sequence (aa 1-18), a specific sequence (aa19-41), a set of collagen repeat sequences (aa42-107) consisting of 22 amino acids, a globular sequence (aa108-244), wherein the globular region is the key site of adiponectin biological activity, and a structure of TNF- α, adiponectin is highly homologous to collagen 1q, a monocolonal and monocolonal adiponectin, and monocolonal or intracorporeal adiponectin, and a structural homology with collagen receptor subunit C, which is known as a receptor for the same as adiponectin, which is secreted by the extracellular domain of adiponectin, collagen receptor subunit C2, adiponectin, a protein secreted from the family of the extracellular domain of the rat, adiponectin, or chimeric protein receptor subunit C2, and/or its receptor subunit type C, and/or its receptor subunit C9, and its metabolic domain, and/or its receptor subunit C, which is known as well as a protein receptor subunit C, and can be involved in the extracellular domain which is involved in the extracellular domain of the extracellular.
The adiponectin is composed of adiponectin monomers, the adiponectin monomers are connected to form trimers, 4-6 trimers are combined to form a high molecular structure, the adiponectin monomers only exist in fat cells, and the adiponectin monomers can be secreted into blood plasma after the polymers are formed.
To date, 3 adiponectin receptors have been discovered, adiponectin receptor 1, adiponectin receptor 2, and T-cadherin, respectively. Among them, Yamau-chi et al found 2 adiponectin receptors, and named adiponectin receptor 1 and adiponectin receptor 2, respectively. The adiponectin receptor has 7 transmembrane domains, but is different from the G protein coupled receptor family structure in that the N terminal is positioned in the membrane, and the C terminal is positioned outside the membrane and can be combined with the adiponectin receptor. Adiponectin receptor 1 is mainly expressed in skeletal muscle cells, is a high-affinity receptor for globular adiponectin and a low-affinity receptor for full-length adiponectin, and is related to adenosine kinase (AMPK); while adiponectin receptor 2 is expressed primarily in hepatocytes, and is a moderate affinity receptor for full-length adiponectin and globular adiponectin, and can activate PPARs. T-cadherin is expressed primarily in endothelial cells and smooth muscle and is primarily responsible for binding to adiponectin in tissues such as heart, muscle, and blood vessels. Parker-Duffen et al found that adiponectin levels in blood of T-cadherin knockout mice were 4 times higher than those of normal mice, and T cadherin expression in heart and blood vessels was reduced, and that T-cadherin expression on cell surfaces could be restored to normal levels after recombinant adiponectin treatment. Because adiponectin plays a variety of biological roles after binding to its receptor, any factor that affects adiponectin receptor expression will affect the exertion of adiponectin biological effects and the occurrence and development of diseases associated with it.
At present, methods for detecting adiponectin mainly include chemiluminescence methods and latex immunoturbidimetry methods. Among them, chemiluminescence is a sensitive and simple method, but has poor selectivity and reacts to a series of compounds, rather than to a single compound. The latex enhanced immunoturbidimetry has the advantages of rapidness, simplicity, convenience, quantitative determination, high precision, easy automation and suitability for simultaneous detection of mass samples. Along with this, a large number of adiponectin ADPN latex enhanced immunoturbidimetry detection kits are available. However, the existing latex-enhanced immunoturbidimetry detection kit has limited detection sensitivity and still cannot meet the requirements of clinical application. Therefore, a kit for detecting adiponectin ADPN latex by using an enhanced immunoturbidimetry method with better sensitivity is urgently needed at present.
Disclosure of Invention
The invention aims to provide a latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN with better sensitivity and a preparation method and an application method thereof.
The invention adopts the following technical scheme to solve the technical problems:
a latex enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN comprises an adiponectin R1 reagent and an adiponectin R2 reagent;
the adiponectin R1 reagent comprises the following components in parts by weight: the buffer solution A is 20-200 mM, the protective agent A is 1-10 mg/mL, the reaction reinforcing agent is 1-5%, the preservative is 0.03-0.2%, and the solvent is purified water;
the adiponectin R2 reagent comprises the following components in parts by weight: 20-200 mM of buffer solution B, 1-10 mg/mL of protective agent B, 0.03-0.2% of preservative, 0.04-0.16% of sensitized polystyrene latex particles coated with anti-human adiponectin antibody, and purified water as solvent; the anti-human adiponectin antibodies in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibodies are connected with the sensitized polystyrene latex particles through a streptavidin-biotin system. Among them, the streptavidin-biotin system can greatly improve the sensitivity of the detection method without increasing non-specific interference. In addition, the streptavidin-biotin combination is stable and cannot be influenced by high dilution of the reaction reagent, so that the accuracy of the detection result is ensured.
In a preferred embodiment of the present invention, in the adiponectin R1 reagent, the buffer a is specifically one or more of a borate buffer, a carbonate buffer, a Tris-HCl buffer, a phosphate buffer and a glycine buffer, and has a pH of 6 to 10; the protective agent A is one or more of bovine serum albumin, ovalbumin and gelatin; the reaction enhancer is one or more of polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000 and polyethylene glycol 20000; the antiseptic is one or more of sodium azide, thimerosal and Proclin 300.
In the technical scheme, Bovine Serum Albumin (BSA), Ovalbumin (OVA) and gelatin can protect the cross-linked antibody on the surface of the latex particles. Polyethylene glycol PEG is a non-ionic water-soluble polymer, has strong hydrophilicity, can destroy electron clouds and hydration layers around proteins in a solution, and promotes specific antigen and antibody molecules to approach and combine to form a macromolecular complex.
In a preferred embodiment of the present invention, buffer A in the adiponectin R1 reagent is preferably Tris-HCl buffer or borate buffer, and has a concentration of 50mM, pH 8.0; the protective agent A is preferably bovine serum albumin with the concentration of 4 mg/mL; the reaction enhancer is preferably polyethylene glycol 6000, and the mass volume ratio is 5%; the preservative is preferably sodium azide with the mass volume ratio of 0.1 percent.
In a preferred embodiment of the present invention, in the adiponectin R2 reagent, the buffer B is specifically one or more of a borate buffer, a carbonate buffer, a Tris-HCl buffer, a phosphate buffer and a glycine buffer, and has a pH of 7 to 9; the protective agent B is one or two of bovine serum albumin and ovalbumin; the antiseptic is one or more of sodium azide, thimerosal and Proclin 300; the anti-human adiponectin antibody in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody is specifically one of a polyclonal antibody and a monoclonal antibody.
In a preferred embodiment of the present invention, in the adiponectin R2 reagent, the buffer B is preferably glycine buffer or Tris-HCl buffer, at a concentration of 60mM, pH 8.0; the protective agent B is preferably bovine serum albumin with the concentration of 5 mg/mL; the preservative is preferably sodium azide with the mass-volume ratio of 0.1%; the final concentration of the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody in the mass-volume ratio is 0.1%; the anti-human adiponectin antibody in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody is preferably a polyclonal antibody.
In a preferred embodiment of the present invention, the anti-human adiponectin antibody in the aforementioned sensitized polystyrene latex particle of an anti-human adiponectin antibody is specifically a murine, rabbit or goat antibody, and is preferably a goat antibody.
In a preferred embodiment of the present invention, the anti-human adiponectin antibody-coated sensitized polystyrene latex particles in the adiponectin R2 reagent are prepared by preparing a biotinylated anti-human adiponectin antibody and streptavidin-coated polystyrene latex particles, respectively, and incubating the biotinylated anti-human adiponectin antibody and the streptavidin-coated polystyrene latex particles in a mixed manner; in the preparation of the biotinylated anti-human adiponectin antibody, the molar ratio of the anti-human adiponectin antibody to biotin is 1: (1 to 100), preferably 1: 10.
in a preferred embodiment of the present invention, the sensitized polystyrene latex particles in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody are specifically carboxylated polystyrene latex particles or aminated polystyrene latex particles, and preferably carboxylated polystyrene latex particles. Wherein, the carboxylated polystyrene latex particles are activated by carbodiimide and then are coated by streptavidin; activating the aminated polystyrene latex particles by glutaraldehyde and then coating by streptavidin; in addition, the particle size of the latex particles is 40-500 nm, preferably 200 nm.
In a preferred embodiment of the present invention, the kit further comprises an adiponectin calibrator;
the adiponectin calibrator comprises the following components in parts by weight: 20-200 mM of buffer solution C, 0.5-8 mg/L of protective agent C, 0.03-0.2% of preservative, 0.2-100 mg/L of adiponectin recombinant protein and purified water as solvent.
In a preferable mode of the invention, in the adiponectin calibrator, the buffer C is one or more of borate buffer, carbonate buffer, Tris-HCl buffer, phosphate buffer and glycine buffer, and the pH value is 7-9; the protectant C is bovine serum albumin; the antiseptic is one or more of sodium azide, thimerosal and Proclin 300.
In a preferred embodiment of the present invention, the adiponectin calibrator is preferably prepared such that the buffer C is glycine buffer or Tris-HCl buffer, has a concentration of 60mM, and has a pH of 8.0; the protective agent C is preferably bovine serum albumin with the concentration of 2 mg/mL; the preservative is preferably sodium azide with the mass-volume ratio of 0.1%; the adiponectin recombinant protein is a commercial product, and the concentration is preferably 40 mg/L.
A preparation method of the latex enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN comprises the following steps:
(1) preparation of adiponectin R1 reagent:
according to the component content of the adiponectin R1 reagent, mixing the component substances in the same container, and uniformly mixing to obtain the adiponectin R1 reagent;
(2) preparation of adiponectin R2 reagent:
① activation and washing of latex particles
Activating a commercial polystyrene latex solution, centrifuging, discarding a supernatant, and repeatedly washing a precipitate for 2-4 times by using a washing buffer solution; resuspending the last pellet in the wash buffer and bringing the final concentration of latex particles to 1% by mass/volume;
② streptavidin-coated latex particles
a. Taking 1mL of the activated and washed polystyrene latex solution, and adding streptavidin to ensure that the final concentration of the streptavidin is 0.1 mg/mL; after mixing, coating the mixture on a shaking table at the temperature of 4-37 ℃ for 0.5-18 h at the rotating speed of 220 rpm;
b. washing streptavidin which is not combined with the latex particles by using PBS buffer solution, and repeating for 2-4 times;
c. and (3) sealing: dissolving the washed latex particles coated with the streptavidin in a buffer solution B containing a protective agent B to ensure that the final concentration of the latex particles in a mass-volume ratio is 0.1-0.3%;
③ biotinylation of anti-human adiponectin antibodies
Selecting a commercial activated product as biotin, mixing the biotin with an anti-human adiponectin antibody, reacting for l-4 h at room temperature, centrifuging, removing a small amount of precipitate, filling the supernatant into a dialysis bag, and dialyzing in PBS buffer solution at 0-4 ℃ overnight; wherein the molar ratio of the anti-human adiponectin antibody to the biotin is 1: (1-100);
④ attachment of biotinylated adiponectin antibodies to streptavidin-coated latex particles
Adding the biotinylated anti-human adiponectin antibody obtained in the step ③ into the streptavidin-coated latex particle solution obtained in the step ②, uniformly mixing, and then incubating for 0.5-2 h in a shaking table at 37 ℃ to form a latex particle-streptavidin-biotin-anti-human adiponectin antibody compound;
⑤ cleaning
And (3) centrifuging the latex particle-streptavidin-biotin-anti-human adiponectin antibody compound solution formed in the step ④, discarding the supernatant, repeatedly washing for 2-4 times, and dissolving the last precipitate in a buffer solution B containing a protective agent B and a preservative to ensure that the mass-volume ratio of the latex particles is 0.04-0.16% in final concentration.
The biotin in the technical scheme is generally selected from activated products, the biotin labeling reaction of the antibody is mild, the activity of the antibody is rarely inhibited, the labeled antibody is stable, the labeled antibody can be combined by streptavidin, the background level is low, and the combination is tight and rapid. The covalent binding of biotin and antibody is usually biotin acylation reaction, and biotin-N-hydroxy-succinimide ester (BNHS) is generated by condensation of biotin and N-hydroxysuccinimide under the action of carbodiimide. the-C-0 group in the ester bond of the BNHS molecule can form a peptide bond with the amino group of lysine in the antibody molecule, so that the antibody is labeled with biotin. Biotin has a small molecular weight, and when biotin-labeled conjugates are formed by reaction with antibodies or enzymes, interference may occur in the binding between biotin and streptavidin and in the application of the system due to steric hindrance of macromolecular proteins. At the moment, a certain number of groups are connected on a side chain of a biotin molecule to form a connecting arm, so that the distance between biotin and a marked macromolecule is increased (such as the biotin is activated by a long arm), the steric effect is reduced, and the sensitivity and the specificity of detection are increased.
In a preferred embodiment of the present invention, in step ①, the washing buffer is specifically one of PBS buffer and MES buffer.
In a preferred embodiment of the present invention, the antibody is labeled with biotin in a suitable ratio of activated biotin to the antibody to be labeled so that the number of biotin molecules labeled per antibody molecule is controlled within a certain range, and after binding to biotin, the activity of the antibody may be reduced, which often occurs when the free amino group necessary for maintaining the activity of the antibody binds to biotin excessively large amounts, and at this time, the biotinylation may reduce or destroy the antigen-binding ability of the antibody, and the molar ratio of the anti-human adiponectin antibody to the biotin is preferably 1: 10 in step ③ of preparing the biotinylated ADPN antibody.
The latex-enhanced turbidimetric immunoassay kit for quantitatively detecting adiponectin ADPN is used on a semi-automatic biochemical analyzer and a full-automatic biochemical analyzer and a nephelometric turbidimetric analyzer to quantitatively detect the adiponectin content in human blood; wherein the blood includes whole blood, serum and plasma.
As one of the preferable modes of the invention, when the latex enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN is used on a semi-automatic and full-automatic biochemical analyzer and is used for detecting adiponectin content, serum or plasma samples are specifically adopted for detection; when the latex enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN is used on a nephelometric turbidimetric analyzer and is used for detecting adiponectin content, whole blood, serum and plasma samples are specifically adopted for detection.
Compared with the prior art, the invention has the advantages that:
(1) the kit further adopts a streptavidin-biotin amplification system on the basis of a latex enhanced immunoturbidimetry principle, so that the detection sensitivity of a turbidimetric reagent is greatly improved; the detection concentration range of the kit reaches 0.3-40.0mg/L, and the kit is rapid, accurate, high in sensitivity and strong in specificity;
(2) the kit can be used on a nephelometry analyzer, and realizes flexible and rapid quantitative detection of multiple sample types; the adiponectin ADPN detection sold in the market mostly adopts serum or plasma for detection, and cannot meet the clinical rapid detection requirement; in addition, the time of about half an hour is required for the detection of the commercially available ADPN, and the kit only needs 5-10 minutes from the sample collection to the fastest detection result, so that more precious time is won for the patient, and the dynamic detection of the patient's condition can be realized;
(3) the kit and the imported kit have good result correlation, no significant difference and accurate and reliable detection results after statistical analysis on the ADPN content detection results of the same sample, can replace imported products in clinic, and greatly reduce the detection cost.
Drawings
FIG. 1 is a calibration graph of the kit of the present invention in example 7;
FIG. 2 is a graph of the linear range of the kit of the invention in example 8;
FIG. 3 is a graph showing the correlation between the kit of the present invention and the marketed kit product in example 8.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN of the embodiment comprises an adiponectin R1 reagent, an adiponectin R2 reagent and an adiponectin calibrator.
The adiponectin R1 reagent comprises the following components in parts by weight: 20mM of buffer solution A (carbonate buffer solution, pH 6.0), 1mg/mL of protective agent A (ovalbumin), 1% of reaction enhancer (polyethylene glycol 4000), 0.03% of preservative (thimerosal) and purified water as solvent.
The adiponectin R2 reagent comprises the following components in parts by weight: 20mM of buffer solution B (borate buffer solution, pH 7.0), 1mg/mL of protective agent B (bovine serum albumin), 0.03% of preservative (thimerosal), 0.04% of sensitized polystyrene latex particles coated with the anti-human adiponectin antibody, and purified water as a solvent.
The adiponectin calibrator comprises the following components in parts by weight: 20mM of buffer C (borate buffer solution, pH 7), 0.5mg/L of protective agent C (bovine serum albumin), 0.03% of preservative (thimerosal), 0.2mg/L of adiponectin recombinant protein and purified water as solvent.
Further, the anti-human adiponectin antibody in the sensitized polystyrene latex particle coated with the anti-human adiponectin antibody is connected with the sensitized polystyrene latex particle through a streptavidin-biotin system; the streptavidin-biotin system can greatly improve the sensitivity of the detection method without increasing non-specific interference. In addition, the streptavidin-biotin combination is stable and cannot be influenced by high dilution of the reaction reagent, so that the accuracy of the detection result is ensured.
Further, in the adiponectin R2 reagent, the anti-human adiponectin antibody in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody is a polyclonal antibody; the anti-human adiponectin antibody is specifically a murine antibody.
Further, the sensitized polystyrene latex particles in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibodies are specifically carboxylated polystyrene latex particles. Wherein, the carboxylated polystyrene latex particles are activated by carbodiimide and then coated by streptavidin, and the particle size range of the latex particles is 40 nm.
Example 2
The latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN of the embodiment comprises an adiponectin R1 reagent, an adiponectin R2 reagent and an adiponectin calibrator.
The adiponectin R1 reagent comprises the following components in parts by weight: 200mM of buffer A (phosphate buffer or glycine buffer, pH 10), 10mg/mL of protective agent A (gelatin), 4% of reaction enhancer (polyethylene glycol 8000 or polyethylene glycol 20000), 0.2% of preservative (Proclin300) and purified water as solvent.
The adiponectin R2 reagent comprises the following components in parts by weight: buffer B (carbonate buffer or phosphate buffer, pH 9;) 200mM, protective agent B (ovalbumin) 10mg/mL, preservative (Proclin300) 0.2%, sensitized polystyrene latex particles coated with anti-human adiponectin antibody 0.16%, and solvent purified water.
The adiponectin calibrator comprises the following components in parts by weight: 200mM of buffer C (carbonate buffer or phosphate buffer, pH 9.0), 8mg/L of protective agent C (bovine serum albumin), 0.2% of preservative (Proclin300), 100mg/L of adiponectin recombinant protein and purified water as solvent.
Further, the anti-human adiponectin antibody in the sensitized polystyrene latex particle coated with the anti-human adiponectin antibody is connected with the sensitized polystyrene latex particle through a streptavidin-biotin system; the streptavidin-biotin system can greatly improve the sensitivity of the detection method without increasing non-specific interference. In addition, the streptavidin-biotin combination is stable and cannot be influenced by high dilution of the reaction reagent, so that the accuracy of the detection result is ensured.
Further, in the adiponectin R2 reagent, the anti-human adiponectin antibody in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody is a monoclonal antibody; the anti-human adiponectin antibody is specifically a rabbit-derived antibody.
Further, the sensitized polystyrene latex particles in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibodies are specifically aminated polystyrene latex particles. Wherein, the aminated polystyrene latex particles are activated by glutaraldehyde and then are coated by streptavidin, and the particle size range of the latex particles is 500 nm.
Example 3
The latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN of the embodiment comprises an adiponectin R1 reagent, an adiponectin R2 reagent and an adiponectin calibrator.
The adiponectin R1 reagent comprises the following components in parts by weight: 50mM of buffer solution A (borate buffer solution or Tris-HCl buffer solution with the pH value of 8.0), 4mg/mL of protective agent A (bovine serum albumin), 5% of reaction enhancer (polyethylene glycol 6000), 0.1% of preservative (sodium azide) and purified water as solvent.
The adiponectin R2 reagent comprises the following components in parts by weight: 60mM of buffer solution B (Tris-HCl buffer solution or glycine buffer solution, pH 8.0), 5mg/mL of protective agent B (bovine serum albumin), 0.1% of preservative (sodium azide), 0.1% of sensitized polystyrene latex particles coated with anti-human adiponectin antibody, and purified water as solvent.
The adiponectin calibrator comprises the following components in parts by weight: 60mM of buffer C (Tris-HCl buffer or glycine buffer, pH 8.0), 2mg/L of protective agent C (bovine serum albumin), 0.1% of preservative (sodium azide), 40mg/L of adiponectin recombinant protein (a commercially available product), and the solvent is purified water.
Further, the anti-human adiponectin antibody in the sensitized polystyrene latex particle coated with the anti-human adiponectin antibody is connected with the sensitized polystyrene latex particle through a streptavidin-biotin system; the streptavidin-biotin system can greatly improve the sensitivity of the detection method without increasing non-specific interference. In addition, the streptavidin-biotin combination is stable and cannot be influenced by high dilution of the reaction reagent, so that the accuracy of the detection result is ensured.
Further, in the adiponectin R2 reagent, the anti-human adiponectin antibody in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody is a polyclonal antibody; the anti-human adiponectin antibody is specifically a goat-derived antibody.
Further, the sensitized polystyrene latex particles in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibodies are specifically carboxylated polystyrene latex particles. Wherein, the carboxylated polystyrene latex particles are activated by carbodiimide and then coated by streptavidin, and the particle size range of the latex particles is 200 nm.
Example 4
The preparation method of the latex-enhanced immunoturbidimetric assay kit for quantitatively detecting adiponectin ADPN in the above embodiment of the present embodiment comprises the following steps:
(1) preparation of adiponectin R1 reagent:
according to the component content of the adiponectin R1 reagent, the components are mixed in the same container, and after uniform mixing, the adiponectin R1 reagent is prepared.
(2) Preparation of adiponectin R2 reagent:
① activation and washing of latex particles
Activating a commercial polystyrene latex solution, centrifuging, discarding the supernatant, and repeatedly washing the precipitate for 2 times by using a washing buffer solution; resuspending the last pellet in the wash buffer and bringing the final concentration of latex particles to 1% by mass/volume; wherein the washing buffer solution is PBS buffer solution;
② streptavidin-coated latex particles
a. Taking 1mL of the activated and washed polystyrene latex solution, and adding streptavidin to ensure that the final concentration of the streptavidin is 0.1 mg/mL; after mixing uniformly, coating the mixture for 0.5h on a shaking table at the rotating speed of 220rpm at the temperature of 4 ℃;
b. washing streptavidin which is not combined with the latex particles by PBS buffer solution (0.1M, pH7.4) and repeating for 2-4 times;
c. and (3) sealing: dissolving the washed latex particles coated with the streptavidin in a buffer solution B containing a protective agent B to ensure that the mass-to-volume ratio of the latex particles is 0.1% (w/v);
③ biotinylation of anti-human adiponectin antibodies
Selecting a commercial activated product as biotin, mixing the biotin with an anti-human adiponectin antibody, reacting for lh at room temperature, centrifuging, and removing a small amount of precipitate; the supernatant was put into a dialysis bag and dialyzed overnight at 0 ℃ in PBS buffer (0.1M, pH 7.4); wherein the molar ratio of the anti-human adiponectin antibody to the biotin is 1: 1;
④ attachment of biotinylated adiponectin antibodies to streptavidin-coated latex particles
Adding 400uL of the biotinylated anti-human adiponectin antibody obtained in the step ③ into 1mL of the streptavidin-coated latex particle solution obtained in the step ②, uniformly mixing, and then incubating for 0.5h in a shaking table at 37 ℃ to form a latex particle-streptavidin-biotin-anti-human adiponectin antibody complex;
⑤ cleaning
The latex particle-streptavidin-biotin-anti-human adiponectin antibody complex solution formed in step ④ was centrifuged, the supernatant was discarded, washing was repeated 2 times, and the last precipitate was dissolved in buffer B containing a protecting agent B and a preservative so that the final concentration of latex particles was 0.04% by mass/volume.
(3) Formulating adiponectin calibrator
The commercially available recombinant human adiponectin protein was dissolved in a solution similar to human serum matrix (i.e., the adiponectin calibrator formulation of the above example except for the recombinant adiponectin protein) to make different concentrations of calibrator. Taking an imported Roche adiponectin calibrator as an original standard, respectively detecting the calibrator with different concentrations for 20 times by using the adiponectin kit, and calculating the average value to obtain the concentration of the adiponectin calibrator: 0.2, L, 3, 10, 30 and 100 mg/L.
Example 5
The preparation method of the latex-enhanced immunoturbidimetric assay kit for quantitatively detecting adiponectin ADPN in the above embodiment of the present embodiment comprises the following steps:
(1) preparation of adiponectin R1 reagent:
according to the component content of the adiponectin R1 reagent, the components are mixed in the same container, and after uniform mixing, the adiponectin R1 reagent is prepared.
(2) Preparation of adiponectin R2 reagent:
① activation and washing of latex particles
Activating a commercial polystyrene latex solution, centrifuging, discarding the supernatant, and repeatedly washing the precipitate for 4 times by using a washing buffer solution; resuspending the last pellet in the wash buffer and bringing the final concentration of latex particles to 1% by mass/volume; wherein the washing buffer solution is specifically PBS buffer solution;
② streptavidin-coated latex particles
a. Taking 1mL of the activated and washed polystyrene latex solution, and adding streptavidin to ensure that the final concentration of the streptavidin is 0.1 mg/mL; after mixing uniformly, coating for 18h on a 37 ℃ shaking table at the rotating speed of 220 rpm;
b. washing streptavidin which is not combined with the latex particles with PBS buffer solution (0.1M, pH7.4) for 2-4 times;
c. and (3) sealing: dissolving the washed latex particles coated with the streptavidin in a buffer solution B containing a protective agent B to ensure that the mass-to-volume ratio of the latex particles is 0.3% (w/v);
③ biotinylation of anti-human adiponectin antibodies
Selecting a commercial activated product as biotin, mixing the biotin with an anti-human adiponectin antibody, reacting for 4 hours at room temperature, centrifuging, and removing a small amount of precipitate; the supernatant was put into a dialysis bag and dialyzed overnight at 4 ℃ in PBS buffer (0.1M, pH 7.4); wherein the molar ratio of the anti-human adiponectin antibody to the biotin is 1: 100, respectively;
④ attachment of biotinylated adiponectin antibodies to streptavidin-coated latex particles
Adding 400uL of the biotinylated anti-human adiponectin antibody obtained in the step ③ into 1mL of the streptavidin-coated latex particle solution obtained in the step ②, uniformly mixing, and then incubating for 2h in a shaking table at 37 ℃ to form a latex particle-streptavidin-biotin-anti-human adiponectin antibody complex;
⑤ cleaning
The latex particle-streptavidin-biotin-anti-human adiponectin antibody complex solution formed in step ④ was centrifuged, the supernatant was discarded, washing was repeated 4 times, and the last precipitate was dissolved in buffer B containing a protecting agent B and a preservative so that the final concentration of latex particles was 0.16% by mass/volume.
(4) Formulating adiponectin calibrator
The commercially available recombinant human adiponectin protein was dissolved in a solution similar to human serum matrix (i.e., the adiponectin calibrator formulation of the above example except for the recombinant adiponectin protein) to make different concentrations of calibrator. Taking an imported Roche adiponectin calibrator as an original standard, respectively detecting the calibrator with different concentrations for 20 times by using the adiponectin kit, and calculating the average value to obtain the concentration of the adiponectin calibrator: 0.2, L, 3, 10, 30 and 100 mg/L.
Example 6
The preparation method of the latex-enhanced immunoturbidimetric assay kit for quantitatively detecting adiponectin ADPN in the above embodiment of the present embodiment comprises the following steps:
(1) preparation of adiponectin R1 reagent:
according to the component content of the adiponectin R1 reagent, the components are mixed in the same container, and after uniform mixing, the adiponectin R1 reagent is prepared.
(2) Preparation of adiponectin R2 reagent:
① activation and washing of latex particles
Activating a commercial polystyrene latex solution, centrifuging, discarding the supernatant, and repeatedly washing the precipitate for 3 times by using a washing buffer solution; resuspending the last pellet in the wash buffer and bringing the final concentration of latex particles to 1% by mass/volume; wherein the washing buffer is MES buffer (50mM, pH 6.5);
② streptavidin-coated latex particles
a. Taking 1mL of the activated and washed polystyrene latex solution, and adding streptavidin to ensure that the final concentration of the streptavidin is 0.1 mg/mL; uniformly mixing, and coating for 16h in a 30 ℃ shaking table at the rotating speed of 220 rpm;
b. washing streptavidin not bound to latex particles with PBS buffer (0.1M, pH7.4), repeated 3 times;
c. and (3) sealing: dissolving the washed latex particles coated with the streptavidin in a buffer solution B containing a protective agent B to ensure that the mass-to-volume ratio of the latex particles is 0.2% (w/v);
③ biotinylation of anti-human adiponectin antibodies
Selecting a commercial activated product as biotin, mixing the biotin with an anti-human adiponectin antibody, reacting for 2 hours at room temperature, centrifuging, and removing a small amount of precipitate; the supernatant was put into a dialysis bag and dialyzed overnight at 2 ℃ for 24 hours in PBS buffer (0.1M, pH 7.4); wherein the molar ratio of the anti-human adiponectin antibody to the biotin is 1: 10;
④ attachment of biotinylated adiponectin antibodies to streptavidin-coated latex particles
Adding 400uL of the biotinylated anti-human adiponectin antibody obtained in the step ③ into 1mL of the streptavidin-coated latex particle solution obtained in the step ②, uniformly mixing, and then incubating for 1h in a shaking table at 37 ℃ to form a latex particle-streptavidin-biotin-anti-human adiponectin antibody complex;
⑤ cleaning
The latex particle-streptavidin-biotin-anti-human adiponectin antibody complex solution formed in step ④ was centrifuged, the supernatant was discarded, washing was repeated 3 times, and the last precipitate was dissolved in buffer B containing a protecting agent B and a preservative so that the final concentration of latex particles was 0.1% by mass/volume.
(5) Formulating adiponectin calibrator
The commercially available recombinant human adiponectin protein was dissolved in a solution similar to human serum matrix (i.e., the adiponectin calibrator formulation of the above example except for the recombinant adiponectin protein) to make different concentrations of calibrator. Taking an imported Roche adiponectin calibrator as an original standard, respectively detecting the calibrator with different concentrations for 20 times by using the adiponectin kit, and calculating the average value to obtain the concentration of the adiponectin calibrator: 0.2, L, 3, 10, 30 and 100 mg/L.
Example 7
This example is a detection method of the latex-enhanced immunoturbidimetric immunoassay kit for quantitatively detecting adiponectin ADPN in the above example:
1. biochemical analyzer detection
Taking Hitachi 7080 as an example, measuring the wavelength of 700nm, respectively taking calibrator solutions (3uL) with different concentrations, adding an adiponectin R1 reagent (160uL), uniformly mixing, incubating at 37 ℃ for 5 minutes, then adding an adiponectin R2 reagent (80uL), uniformly mixing, incubating at 37 ℃ for 1 minute, reading absorbance values A1 of each tube, reacting for 4-5 minutes, reading absorbance values A2 of each tube, calculating absorbance differences △ A-A2-A1, repeatedly measuring for 3 times per tube, taking the average value of the absorbance differences △ A measured 3 times of each calibration tube as a vertical coordinate, taking the corresponding calibrator concentration as a horizontal coordinate, and drawing a calibration curve of 'concentration-absorbance difference' (see figure 1).
And (3) taking a serum or plasma sample to be detected, determining the absorbance difference of the sample by the same method, and substituting the absorbance difference into the calibration curve to calculate the content of adiponectin ADPN in the sample to be detected. If the concentration of the adiponectin in the serum or the plasma exceeds the range of the calibration curve, the sample needs to be diluted and then detected so as to ensure the accuracy of the detection result.
The kit is not only suitable for Nigri 7080, but also suitable for semi-automatic and full-automatic biochemical analyzers of other brands and models, and specific parameters can be adjusted according to the instruments.
2. NORMAN series nephelometry detection
Adding 35uL (20 uL of serum or plasma) of whole blood into a sample reaction cup, then adding 240uL of R1 reagent, mixing uniformly, after the scattered light intensity in the reaction cup is stable, adding 80uL of R2 reagent, mixing uniformly, measuring the change difference △ A of the scattered light intensity within 1-2 minutes, and substituting △ A into a calibration curve to calculate the content of adiponectin in the sample to be measured.
Example 8
This example is used to evaluate the latex enhanced immunoturbidimetric kit for the quantitative detection of adiponectin ADPN of the above examples:
(1) linear range
The adiponectin high-concentration samples (40mg/L) close to the upper limit of the linear range are diluted by physiological saline according to 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64 to prepare 6 diluted concentration (xi) solutions, and the concentration of each diluted sample is determined by the biochemical analyzer detection method. The measurement was repeated 3 times for each concentration, and the average value (yi) of the measurement results was obtained. The linear regression equation was calculated using the dilution concentration (xi) as an independent variable and the measurement result average value (yi) as a dependent variable. The correlation coefficient r of the linear regression is calculated according to the formula (1), and the result shows that the regression equation is that y is 1.0086X-0.1464, and the correlation coefficient r is 0.9998, which shows that the kit of the invention has better correlation in the linear range of 0.3mg/L-40mg/L (see figure 2).
Figure BDA0002387155210000201
(2) Sensitivity (lowest detection limit)
The 5% bovine serum albumin solution is used as a blank sample, the measurement is repeated for 20 times according to the detection method of a biochemical analyzer, the lowest detection limit is reported by adding two times of standard deviation to the blank mean value, and the result shows that the lowest detection limit of the kit is 0.3 mg/L.
(3) Repeatability and accuracy
The adiponectin calibrants with the respective identification values of 0.67mg/L and 51.4mg/L of AUDIT company are used as samples, the measurement is carried out according to the detection method of the biochemical analyzer, the measurement is respectively repeated for 10 times for each concentration, and the measurement mean values are respectively calculated
Figure BDA0002387155210000204
And standard deviation (S) of
Figure BDA0002387155210000203
Calculating the coefficient of variation to carry out repeatability investigation, wherein the result shows that the coefficient of variation is 2.86 percent and 1.38 percent respectively; to be provided with
Figure BDA0002387155210000205
Relative deviations were calculated for accuracy studies and were 1.49% and 0.79%, respectively, as shown in table 1. .
TABLE 1 repeatability and accuracy Studies of adiponectin kits
Figure BDA0002387155210000202
Figure BDA0002387155210000211
(4) Difference between batches
Preparing three batches of kits according to the method of the embodiment of the invention, repeatedly measuring the same serum sample by using the three batches of kits, repeatedly measuring each batch number for 3 times, and respectively calculating the mean value of the 3 measurements of each batch
Figure BDA0002387155210000219
(i ═ l,2,3), the relative deviation (R) was calculated according to the formulae (2) and (3), and the result showed a relative deviation R of 2.68%, as shown in table 2.
Figure BDA0002387155210000212
Figure BDA0002387155210000213
In the formula:
Figure BDA0002387155210000214
Figure BDA0002387155210000215
maximum value of (1); in the formula:
Figure BDA0002387155210000216
Figure BDA0002387155210000217
minimum value of (1);
Figure BDA0002387155210000218
mean values of three reagent batches.
TABLE 2 inter-batch Difference study of adiponectin kits
Figure BDA0002387155210000221
(5) Interference experiment
Taking adiponectin calibrator solution with the concentration of 0.67mg/L, adding each interfering substance solution with the same volume respectively to enable the concentration of each added interfering substance to be 10g/L of hemoglobin, 684umol/L of bilirubin and 20g/L of triglyceride, repeatedly measuring each prepared sample by using the kit provided by the invention for 3 times, taking an average value, comparing with a sample added with the same volume of distilled water, and observing the relative deviation of ADPN measured values after the interfering substance is added and the distilled water is added. The results show that the relative deviation of the measured value of the above concentration interfering substance from the measured value after the addition of the same volume of distilled water is not more than 4%. When the interference substances including hemoglobin, bilirubin and triglyceride exist in the detection sample at a certain concentration, the influence on the detection result of the kit is small.
(6) Stability of
The kit of the invention is examined for the stability of the bottle opening and the long-term stability.
The bottle opening stability is as follows: after the kit is unpacked and stored at 2-8 ℃ for 30 days, the adiponectin calibrator with the AUDIT standard values of 0.67mg/L and 51.4mg/L is taken out and determined according to the detection method of the biochemical analyzer, the adiponectin calibrator is repeatedly determined for 3 times according to each concentration, and the relative deviation between the detection result and the standard value is calculated. The result shows that the relative deviation of the detection value and the marked value of the agent box of the invention is 1.99 percent and 1.12 percent respectively after 30 days of bottle opening, and the bottle opening stability is better.
Long-term stability: after the kit is stored at the temperature of 2-8 ℃ for 12 months, the adiponectin calibrator with the AUDIT standard values of 0.24mg/L and 45.9mg/L respectively is taken out to be measured according to the detection method of the biochemical analyzer, each concentration is repeatedly measured for 3 times, and the relative deviation between the detection result and the standard value is calculated. The result shows that the relative deviation of the detection value and the indication value of the kit is 2.78 percent and 1.32 percent respectively, and the kit is relatively stable when being stored for 12 months at the temperature of 2-8 ℃.
See table 3 for details.
TABLE 3 decap and Long-term stability Studies of adiponectin kits
Figure BDA0002387155210000231
(7) Comparison of the kits of the invention with products already on the market
The hospital provided 79 hospitalized sera, 45 males and 34 females, with a mean age of 39. The adiponectin determination kit and the commercially available imported Roche adiponectin electrochemiluminescence kit are used for repeated detection of the samples for 2 times respectively, the average values are calculated respectively, the detection results of 79 samples are subjected to linear regression analysis, and the correlation coefficients of the detection results of the two kits are calculated. The results show that the correlation coefficient r of the detection results of the two kits is 0.9986, and the linear regression equation is 0.9475x +0.1073 (see fig. 3). The adiponectin assay kit of the present invention is in good agreement with the commercially available imported Roche adiponectin electrochemiluminescence kit according to the American society for Clinical Laboratory Standards Institute (CLSI) documentation requirements (r > 0.975). Therefore, the adiponectin determination kit can be used for replacing an imported kit clinically, the detection cost is reduced, and the economic burden of patients is relieved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. A latex enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN is characterized by comprising an adiponectin R1 reagent and an adiponectin R2 reagent;
the adiponectin R1 reagent comprises the following components in parts by weight: the buffer solution A is 20-200 mM, the protective agent A is 1-10 mg/mL, the reaction reinforcing agent is 1-5%, the preservative is 0.03-0.2%, and the solvent is purified water;
the adiponectin R2 reagent comprises the following components in parts by weight: 20-200 mM of buffer solution B, 1-10 mg/mL of protective agent B, 0.03-0.2% of preservative, 0.04-0.16% of sensitized polystyrene latex particles coated with anti-human adiponectin antibody, and purified water as solvent; wherein the anti-human adiponectin antibodies in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibodies are connected with the sensitized polystyrene latex particles through a streptavidin-biotin system.
2. The latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN according to claim 1, wherein in the adiponectin R1 reagent, the buffer solution A is one or more of borate buffer solution, carbonate buffer solution, Tris-HCl buffer solution, phosphate buffer solution and glycine buffer solution, and the pH is 6-10; the protective agent A is one or more of bovine serum albumin, ovalbumin and gelatin; the reaction enhancer is one or more of polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000 and polyethylene glycol 20000; the antiseptic is one or more of sodium azide, thimerosal and Proclin 300.
3. The latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN according to claim 1, wherein in the adiponectin R2 reagent, the buffer B is one or more of borate buffer, carbonate buffer, Tris-HCl buffer, phosphate buffer and glycine buffer, and the pH is 7-9; the protective agent B is one or two of bovine serum albumin and ovalbumin; the antiseptic is one or more of sodium azide, thimerosal and Proclin 300; the anti-human adiponectin antibody in the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody is specifically one of a polyclonal antibody and a monoclonal antibody.
4. The latex-enhanced turbidimetric immunoassay kit for quantitatively detecting adiponectin ADPN according to claim 1, wherein the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody in the adiponectin R2 reagent are prepared by preparing a biotinylated anti-human adiponectin antibody and streptavidin-coated polystyrene latex particles, respectively, and mixing and incubating the biotinylated anti-human adiponectin antibody and the streptavidin-coated polystyrene latex particles; in the preparation of the biotinylated anti-human adiponectin antibody, the molar ratio of the anti-human adiponectin antibody to biotin is 1: (1-100).
5. The latex-enhanced turbidimetric immunoassay kit for quantitatively detecting adiponectin ADPN according to claim 1, wherein the sensitized polystyrene latex particles among the sensitized polystyrene latex particles coated with the anti-human adiponectin antibody are specifically carboxylated polystyrene latex particles or aminated polystyrene latex particles; wherein, the carboxylated polystyrene latex particles are activated by carbodiimide and then are coated by streptavidin; activating the aminated polystyrene latex particles by glutaraldehyde and then coating by streptavidin; the particle size range of the latex particles is 40-500 nm.
6. The latex-enhanced immunoturbidimetry kit for quantitatively detecting adiponectin ADPN according to any one of claims 1 to 5, further comprising an adiponectin calibrator;
the adiponectin calibrator comprises the following components in parts by weight: 20-200 mM of buffer solution C, 0.5-8 mg/L of protective agent C, 0.03-0.2% of preservative, 0.2-100 mg/L of adiponectin recombinant protein and purified water as solvent.
7. The latex-enhanced immunoturbidimetric kit for quantitatively detecting adiponectin ADPN according to claim 6, wherein in the adiponectin calibrator, the buffer C is one or more of borate buffer, carbonate buffer, Tris-HCl buffer, phosphate buffer and glycine buffer, and the pH is 7-9; the protectant C is bovine serum albumin; the antiseptic is one or more of sodium azide, thimerosal and Proclin 300.
8. A method for preparing the latex-enhanced immunoturbidimetric kit for the quantitative determination of adiponectin ADPN as defined in any one of claims 1 to 7, comprising the steps of:
(1) preparation of adiponectin R1 reagent:
according to the component content of the adiponectin R1 reagent, mixing the component substances in the same container, and uniformly mixing to obtain the adiponectin R1 reagent;
(2) preparation of adiponectin R2 reagent:
① activation and washing of latex particles
Activating a polystyrene latex solution, centrifuging, discarding a supernatant, and repeatedly washing the precipitate for 2-4 times by using a washing buffer solution; resuspending the last pellet in the wash buffer and bringing the final concentration of latex particles to 1% by mass/volume;
② streptavidin-coated latex particles
a. Taking 1mL of the activated and washed polystyrene latex solution, and adding streptavidin to ensure that the final concentration of the streptavidin is 0.1 mg/mL; after mixing, coating the mixture on a shaking table at the temperature of 4-37 ℃ for 0.5-18 h at the rotating speed of 220 rpm;
b. washing streptavidin which is not combined with the latex particles by using PBS buffer solution, and repeating for 2-4 times;
c. and (3) sealing: dissolving the washed latex particles coated with the streptavidin in a buffer solution B containing a protective agent B to ensure that the final concentration of the latex particles in a mass-volume ratio is 0.1-0.3%;
③ biotinylation of anti-human adiponectin antibodies
Selecting an activated product as biotin, mixing the biotin with an anti-human adiponectin antibody, reacting for l-4 h at room temperature, centrifuging, removing a small amount of precipitate, filling the supernatant into a dialysis bag, and dialyzing in PBS buffer solution at 0-4 ℃ overnight; wherein the molar ratio of the anti-human adiponectin antibody to the biotin is 1: (1-100);
④ attachment of biotinylated adiponectin antibodies to streptavidin-coated latex particles
Adding the biotinylated anti-human adiponectin antibody obtained in the step ③ into the streptavidin-coated latex particle solution obtained in the step ②, uniformly mixing, and then incubating for 0.5-2 h in a shaking table at 37 ℃ to form a latex particle-streptavidin-biotin-anti-human adiponectin antibody compound;
⑤ cleaning
And (3) centrifuging the latex particle-streptavidin-biotin-anti-human adiponectin antibody compound solution formed in the step ④, discarding the supernatant, repeatedly washing for 2-4 times, and dissolving the last precipitate in a buffer solution B containing a protective agent B and a preservative to ensure that the mass-volume ratio of the latex particles is 0.04-0.16% in final concentration.
9. The method for preparing the latex-enhanced turbidimetric immunoassay kit for the quantitative determination of adiponectin ADPN according to claim 8, wherein the washing buffer in step ① is one of PBS buffer and MES buffer, and the molar ratio of the anti-human adiponectin antibody to the biotin in step ③ is 1: 10.
10. The method for using the latex-enhanced turbidimetric immunoassay kit for quantitatively detecting adiponectin ADPN according to any one of claims 1 to 7, wherein the latex-enhanced turbidimetric immunoassay kit for quantitatively detecting adiponectin ADPN is used on a semi-automatic and full-automatic biochemical analyzer and a nephelometric turbidimetric analyzer to quantitatively detect the adiponectin content in human blood; wherein the blood includes whole blood, serum and plasma.
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