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Method and kit for detecting hepatitis E virus (HEV) antibody and method for preparing kit

A technology for detecting antibodies and kits, which is applied in the field of detection of hepatitis E virus antibodies based on double-labeled time-resolved fluorescence immunoassay, which can solve the problems of high environmental and operator influence, low sensitivity, linearity and stability, and difficulty in automation, etc. problem, to achieve the effect of helping diagnosis and prevention, improving clinical detection rate, and reducing missed detection

Inactive Publication Date: 2014-10-08
GUANGZHOU FENGHUA BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Anti-HEV IgG seroconversion or 4 times or more increase in anti-HEV IgG content, this indicator needs to be quantitatively detected in double serum, which is not conducive to early diagnosis
Due to radioactive pollution, RIA has a great impact on the environment and operators, and the intra-assay and inter-assay variation is large, making it difficult to automate; ELISA uses macromolecular enzyme labels, and the enzyme is easily inactivated. This detection method relies on colorimetry or polarized light. The technology is subject to too many interference factors (even the excellent "tube ELISA", the change of the shape of the test tube will affect the results of the test), and its sensitivity, linearity and stability cannot be higher than that of RIA; the chemiluminescence of CLIA is usually It is completed in an instant, the luminescence peak decays quickly, and the temperature and pH value have a great influence on the luminescence. Such factors affect the application of this type of method; ECLI is still a non-open system, and the reagents rely on imports. The reagents are expensive, maintenance and detection costs High, which also limits its promotion and application

Method used

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  • Method and kit for detecting hepatitis E virus (HEV) antibody and method for preparing kit
  • Method and kit for detecting hepatitis E virus (HEV) antibody and method for preparing kit
  • Method and kit for detecting hepatitis E virus (HEV) antibody and method for preparing kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] An embodiment of the kit for detecting hepatitis E virus antibody according to the present invention, the kit includes: a solid phase carrier coated with HEV recombinant antigen, Eu 3+ Labeled mouse anti-human IgG antibody, Sm 3+ Labeled mouse anti-human μ chain antibody, calibrator, sample diluent, experimental buffer, concentrated washing solution and enhancement solution; the HEV recombinant antigen contains ORF2 and ORF3 epitopes.

[0043] The present invention also provides a preparation method of the above-mentioned test kit for detecting hepatitis E virus antibody, said method comprising the following steps:

[0044] (1) Preparation of antigen-coated solid-phase carrier: Dilute HEV recombinant antigen with coating buffer to 0.1-10 μg / mL, coat on the solid-phase carrier, wash the solid-phase carrier once, and then use a blocking The solution was sealed, the solid-phase carrier was shaken dry, dried in the air, vacuum-packed, and stored at 2-8°C for later use. Pr...

Embodiment 2

[0057] The kit experimental method of the present invention is simple and rapid, and can be operated automatically, and the detection system is an open operating system. The using method of the kit for detecting hepatitis E virus antibody described in the present invention is as follows:

[0058] (1) Reagent preparation

[0059] ① Antigen solid-phase carrier: Equilibrate the reagent and the required amount of antigen solid-phase carrier to room temperature (20-25°C). The rest of the solid-phase antigen carrier was put into a ziplock bag in time to be sealed and stored at 2-8°C.

[0060] ②Washing liquid: Mix 40ml of concentrated washing liquid and 960ml of purified water in a clean container, and use it as a working washing liquid for later use. Please prepare purified water by yourself.

[0061] ③ Marker mixed working solution: Prepare within 30 minutes before use, mix Eu 3+ mouse anti-human IgG antibody, Sm 3+ Mouse anti-human μ chain antibody and experimental buffer by v...

Embodiment 3

[0072] Analytical performance evaluation of the test kit for detecting hepatitis E virus antibody of the present invention:

[0073] (1) Detection performance of HEV IgG antibody

[0074] Conformity rate of negative reference products: 30 national negative reference products for detection of HEV IgG antibody, no more than 1 positive reaction;

[0075] Conformity rate of positive reference products: 10 national positive reference products for detection of HEV IgG antibody, no more than 1 negative reaction;

[0076] Minimum detection limit: 6 copies of the national minimum detection limit reference product, no less than 3 positive reactions and negative reaction of matrix serum S1;

[0077] Linearity: After testing the reference products of the enterprise, after the statistical analysis of the measured values ​​of 5 samples from L1 to L5, the linear correlation coefficient r between the measured value and the theoretical value is greater than 0.98, such as image 3 .

[0078]...

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Abstract

The invention discloses a method and a kit for detecting a hepatitis E virus (HEV) antibody and a method for preparing the kit. With adoption of double-tagging time-resolved fluoroimmunoassay technology, the problem that the previous HEV IgG antibodies and IgM antibodies need to be independently detected or the IgG antibodies and IgM antibodies cannot be distinguished during combined detection is solved; trace detection of the HEV IgG antibodies and IgM antibodies is realized; with adoption of combined detection of HEV IgG antibodies and IgM antibodies in serum of patients, the clinical detection rate of the HEV can be improved, and disease diagnosis and prevention can be promoted; an antigen of the kit comprises multiple main epitopes, and the detection leakage condition can be reduced to the greatest degree. The kit has the characteristics of high detection result accuracy, high sensitivity, high specificity, high stability, simple and convenient detection method, high efficiency and the like.

Description

technical field [0001] The invention relates to a method and kit for detecting hepatitis E virus antibody, in particular to a method for detecting hepatitis E virus antibody based on double-labeled time-resolved fluorescence immunoassay, a kit and a preparation method of the kit. Background technique [0002] Hepatitis E virus is caused by hepatitis E virus (HEV), and is an intestinally transmitted disease mainly characterized by inflammation and necrosis of liver parenchymal cells. The patients are mainly adults, and the case fatality rate is high, especially for pregnant women in the last 3 months of pregnancy, the case fatality rate can reach 10% to 39%. Hepatitis E virus was first discovered in the Indian subcontinent, and there are reports of greater prevalence in Central Asia, Southeast Asia, Africa, and the Indian subcontinent. Most outbreaks are waterborne. Food-borne reports have also been found in the literature. The infection rate of hepatitis E among Chinese p...

Claims

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Application Information

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IPC IPC(8): G01N33/576
CPCG01N33/56983G01N33/5767
Inventor 谭玉华卢德祥李奕辉范主桥
Owner GUANGZHOU FENGHUA BIOENG
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