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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: 2014-05-07
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

[0044] Example 1 Preparation of anti-human adiponectin polyclonal antibody

[0045] Adiponectin purchased from BIO-RAD in the United States was used as the antigen, and subcutaneous multi-point injection was used for immunization to prepare polyclonal antibodies against human adiponectin.

[0046] 100μg of adiponectin was injected with an equal amount of Freund's complete adjuvant to emulsify the antigen, and the back of the New Zealand white rabbits were injected subcutaneously with 2.2 to 2.5kg, and then every two weeks, the antigen was emulsified with Freund's incomplete adjuvant, and the injection was repeated in the same way Carry out booster immunization 3 times for a total of 4 times. Then 100ml rabbit blood was drawn from the vein and centrifuged to obtain serum. Take 10ml rabbit serum through the immunoaffinity chromatography column, wash the column repeatedly with 1000ml TBS (20mM Tris, pH 7.4, 500mM NaCl, 0.05% Tween-20) buffer, discard the effluent, and use 10ml after...

Embodiment 2

[0047] Example 2: Preparation of latex particles coated with anti-human adiponectin polyclonal antibody

[0048] Disperse or dissolve 10.0g styrene, 2g acrylic acid, and 0.1g sodium dodecylbenzene sulfonate as surfactants in 100ml deionized water, bubbling in nitrogen, slowly adding 0.1g potassium persulfate as initiator, heating The temperature was raised to 80°C, stirred and reacted for about 3 hours, and cooled to obtain latex particles. The measured average particle size range was 90 nm.

[0049] The Fc fragment of anti-human adiponectin polyclonal antibody is coupled to the surface of latex particles by chemical bonding. The specific steps are as follows:

[0050] 1) 100mg of latex particles containing carboxyl groups in 5ml MES buffer (50mM, pH 6.0) or PBS buffer (50mM, pH 7.2), add the surfactant sodium dodecyl sulfonate (final concentration 0.01%), A solution of latex particles is obtained.

[0051] 2) Dissolve the anti-human adiponectin polyclonal antibody in 5ml MES buffer...

Embodiment 3

[0053] Example 3: Preparation of the kit

[0054] R1 reagent: It is made of the following components by weight percentage: 3% sodium chloride, 2.5% mannitol, 5% Tween 80, 0.5% sodium azide, pH 7.4, 250 mmol / L MES buffer liquid.

[0055] R2 reagent: made of the following weight percentage components: 4mg / mL latex particles coated with anti-human adiponectin polyclonal antibody, 3% sodium chloride, 2.5% mannitol, 5% Tween 80 , 0.5% sodium azide, pH 7.4, 250mmol / L MES buffer.

[0056] Preparation of calibrator: Purified natural adiponectin was purchased from BIO-RAD in the United States as the mother liquid, and bovine serum albumin was used to serially dilute the mother liquid to prepare the calibrator: S5: 50mg / L, S4: 25mg / L, S3: 10mg / L, S2: 5mg / L, S1: 1mg / L, S0: purified 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 Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/54393G01N33/545G01N2333/715
Inventor 侯志波
Owner SHENZHEN HONGMED INFAGEN CO LTD
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