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Kit for measuring adiponectin by nano latex enhanced immune turbidimetry

A technology for enhancing immunity and nano-latex, applied in the field of medical immunology in vitro diagnosis, can solve the problems of low degree of automation, long operation time, poor repeatability, etc., and achieve the effects of improving detection sensitivity, increasing sensitivity and preventing flocculation.

Active Publication Date: 2015-04-08
SHENZHEN HONGMED INFAGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the ELISA method has been used clinically for nearly two decades, it still has some fatal shortcomings, such as long operation time and low degree of automation
Radioimmunoassay (RIA) has high sensitivity, but is unstable and has worse repeatability than ELISA, and there is a risk of radioactive contamination

Method used

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  • Kit for measuring adiponectin by nano latex enhanced immune turbidimetry
  • Kit for measuring adiponectin by nano latex enhanced immune turbidimetry
  • Kit for measuring adiponectin by nano latex enhanced immune turbidimetry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 Preparation of anti-human adiponectin polyclonal antibody

[0044] Using adiponectin purchased from BIO-RAD Company of the United States as an antigen, multi-point subcutaneous injection was used for immunization to prepare anti-human adiponectin polyclonal antibody.

[0045] 100 μg of adiponectin was emulsified with the same amount of Freund's complete adjuvant to emulsify the antigen, and injected subcutaneously into 2.2-2.5 kg New Zealand white rabbits at multiple points on the back, and then emulsified the antigen with Freund's incomplete adjuvant every two weeks, and repeated injections in the same way Booster immunization was carried out 3 times, for a total of 4 immunizations. Then 100ml of rabbit blood was extracted from the vein and centrifuged to obtain serum. Take 10ml rabbit serum to pass through the immunoaffinity chromatography column, wash the chromatography column repeatedly with 1000ml TBS (20mM Tris, pH7.4, 500mM NaCl, 0.05%Tween-20) buff...

Embodiment 2

[0046] Example 2: Preparation of latex particles coated with anti-human adiponectin polyclonal antibody

[0047] Disperse or dissolve 10.0g styrene, 2g acrylic acid, and 0.1g sodium dodecylbenzenesulfonate as a surfactant in 100ml deionized water, feed nitrogen, slowly add 0.1g potassium persulfate as an initiator, and heat to 80°C, stirred and reacted for about 3 hours, cooled to obtain latex particles, and the measured average particle size range was 90nm.

[0048] The Fc fragment of anti-human adiponectin polyclonal antibody is coupled to the surface of latex particles by chemical bonding, and the specific steps are as follows:

[0049] 1) 100mg carboxyl-containing latex particles in 5ml MES buffer (50mM, pH6.0) or PBS buffer (50mM, pH7.2), add surfactant sodium dodecylsulfonate (final concentration 0.01% ) to obtain a latex particle solution.

[0050] 2) Dissolve the anti-human adiponectin polyclonal antibody in 5ml MES buffer (50mM, pH6.0) or PBS buffer (50mM, pH7.2) to...

Embodiment 3

[0052] Embodiment 3: the preparation of kit

[0053] R1 reagent: made of the following components by weight: 3% sodium chloride, 2.5% mannitol, 5% Tween 80, 0.5% sodium azide, pH7.4, 250mmol / L MES buffer liquid.

[0054] R2 reagent: made of the following components by weight percentage: 4mg / mL latex particles coated with anti-human adiponectin polyclonal antibody, 3% sodium chloride, 2.5% mannitol, 5% Tween 80 , 0.5% sodium azide, pH7.4, 250mmol / L MES buffer.

[0055] Preparation of calibration products: Purified natural adiponectin was purchased from BIO-RAD Company of the United States as a mother solution, and the mother solution was diluted with bovine serum albumin to prepare calibration products: S5: 50mg / L, S4: 25mg / L, S3: 10mg / L L, S2: 5mg / L, S1: 1mg / L, S0: pure water. The concentration of bovine serum albumin is 100-1000mg / L.

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Abstract

The invention provides a kit for measuring adiponectin by a nano latex enhanced immune turbidimetry. The kit comprises reagent R1, reagent R2 and adiponectin antigen calibration material solution, wherein the reagent R1 comprises electrolyte, a stabilizer, a surface active agent, a preservative and buffer solution; the reagent R2 comprises latex particles coated by anti-human adiponectin polyclonal antibody, electrolyte, a stabilizer, a surface active agent, a preservative and buffer solution; the latex particles are copolymer formed by styrene and acrylic acid; the adiponectin antigen calibration material solution comprises adiponectin and a stabilizer; the surfaces of the latex particles in the reagent R2 have carboxyl groups, and the particle size range of the latex particles is 80-100nm. The kit is simple to operate, rapid and accurate in measurement, high in sensitivity and specificity, and low in detection cost.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis of medical immunity, in particular to a kit for measuring adiponectin in human serum by adopting nano latex-enhanced immune turbidimetry. Background technique [0002] Adiponectin (APN), also known as Acrp30, GBP28 or AdipoQ, is a cytokine synthesized and secreted by adipocytes, which can exist stably in human plasma, with a concentration range of about 3.0-30.0 mg / L, accounting for about 0.01% of protein. Human adiponectin contains 244 amino acids, including 3 domains: an amino-terminal signal sequence, a collagen domain, and a carboxy-terminal globular domain. Adiponectin exists in blood in three isoforms: trimer (65kb), hexamer (150kb) and high molecular weight polymer (18-36 multimers, >280kb). Clinical studies have shown that adiponectin is closely related to obesity, type II diabetes, coronary heart disease, and insulin resistance, and has properties of anti-atherosclerosis, anti-infla...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/54393G01N33/545G01N2333/715
Inventor 侯志波
Owner SHENZHEN HONGMED INFAGEN CO LTD
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