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Hen egg drop syndrome virus double-antibody sandwich ELISA detection method

A syndrome virus, double antibody sandwich technology, applied in the field of pathogen detection, can solve the problems of low separation probability, low detection rate, easy to miss detection, etc., and achieve the effect of overcoming cumbersome operations and easy operation.

Inactive Publication Date: 2018-10-02
ZHENGZHOU HOUYI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection methods of the virus include virus isolation and culture, agar diffusion test, neutralization test, PCR detection, etc. The virus isolation diagnosis is relatively reliable, the isolation probability is low and time-consuming and laborious; agar expansion detection is simple and fast, but the detection rate is low , it is easy to miss detection; the operation of neutralization test is complicated, and it takes a long time to make an early diagnosis; the instability and non-specificity of PCR detection is a fact that cannot be ignored

Method used

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  • Hen egg drop syndrome virus double-antibody sandwich ELISA detection method
  • Hen egg drop syndrome virus double-antibody sandwich ELISA detection method
  • Hen egg drop syndrome virus double-antibody sandwich ELISA detection method

Examples

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Embodiment 1

[0044] 1. Experimental method

[0045] 1.1 EDS 76 subculture

[0046]The egg drop syndrome seed virus (AV71) strain was diluted 1:10 times with sterilized normal saline, and double antibody (penicillin-streptomycin solution) was added at 100 units per ml for 15 min, 0.2 mL per embryo, placed in Cultured in a 37°C incubator. Embryos were irradiated twice a day, and the dead embryos within 24 hours were discarded. The chicken embryos that died at 24-96 hours and the live chicken embryos after 96 hours were placed in the refrigerator at 4 °C overnight, and the eggshell and shell membrane at the air cell were removed. The allantoic fluid was collected separately under sterile conditions and stored for later use.

[0047] 1.2 EDS 76 concentration and purification of

[0048] Take the virus supernatant, add solid PEG and NaCl under slow stirring, to the final concentrations of 70 g / L and 23 g / L, respectively, and magnetically stir overnight at 4 °C. The next day, centrifuge at...

Embodiment 2

[0123] Get 200 parts of chicken fresh feces or anus swab samples from Gongyi, Dengfeng, Kaifeng, Xinxiang, and five places in Luoyang around Zhengzhou, and use this invention to detect after being treated with physiological saline, and the method of the present invention detects 35 positives. The yield rate was 17.5%; 7 samples had hemagglutination value by hemagglutination test; 21 samples ran out of positive bands by PCR method. It shows that the double-antibody sandwich method ELISA of the invention detects egg drop syndrome virus, has high sensitivity and good specificity, and can be used for clinical diagnosis.

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Abstract

The invention discloses a hen egg drop syndrome virus double-antibody sandwich ELISA detection method, specifically comprising the following steps: (1) immunizing a laying hen and a male healthy adultrabbit by using EDS76 as an antigen for preparing chicken anti-EDS76 serum and rabbit anti-EDS76 serum; (2) purifying anti-EDS76 immune globulin in the chicken anti-EDS76 serum and the rabbit anti-EDS76 serum to prepare chicken anti-EDS76 immune globulin and rabbit anti-EDS76 immune globulin; (3) performing detection by ELISA; (4) determining a critical value: take X+3S being equal to 0.331 as the determined critical value. The method provided by the invention overcomes the disadvantages of the traditional method that operation is cumbersome, time-consuming and labor-consuming, the detectionrate is low and the like, and solves the problem of persistent leaked detection of infected animals caused by the fact that only the antibody is checked but the virus is not checked. The results of specificity, crossover trials and repetitive tests show that the method is a specific, sensitive, rapid and easy-to-operate method.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection, and in particular relates to a double-antibody sandwich ELISA detection method for chicken egg drop syndrome virus. Background technique [0002] Egg drop syndrome virus (EDS) 76 ) belongs to the genus Adenoviridae Adenothymovirus, which can clinically cause Egg Drop Syndrome (Egg Drop Syndrome) in chickens, ducks, geese and other poultry, so it is also called Egg Drop Syndrome Virus or EDS virus. Egg drop syndrome was first reported by Dutch scholar Van Eck in 1976, so it is also called EDS 76 Virus. The pathogen was isolated in 1977, and now it has developed and isolated in most countries in the world such as Britain, France, West Germany, Italy, Belgium, Spain, Denmark, Sweden, Norway, the United States, Japan, and India. [0003] EDS 76 The virus is the only member of subgroup III avian adenovirus, although there are some differences in the restriction endonuclease map analysis...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 魏志刚许贤会梁桂霞
Owner ZHENGZHOU HOUYI PHARMA
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