Deer epidemic hemorrhage competitive enzyme-linked immunosorbent assay kit and preparation method and use thereof

A deer epidemic hemorrhage and enzyme-linked immunosorbent assay technology, applied in the biological field, can solve problems such as economic loss of EHDV, and achieve the effect of high sensitivity and strong specificity

Inactive Publication Date: 2010-03-17
花群义
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  • Abstract
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AI Technical Summary

Problems solved by technology

At present, there is no effective vaccine and treatment for this disease. It will take a lot of manpower and material resources to control and eliminate EHD, which will bring great economic losses to the countries where EHDV is endemic.

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  • Deer epidemic hemorrhage competitive enzyme-linked immunosorbent assay kit and preparation method and use thereof

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Embodiment Construction

[0046] The preparation method of the deer epidemic hemorrhagic disease virus antibody competition enzyme-linked immunosorbent assay kit of the present invention includes but is not limited to the following examples. The concrete steps of this method are:

[0047] 1. Preparation of deer epidemic hemorrhagic disease virus ELISA coated antigen: BHK-21 cells grow into a single layer and inoculate with deer epidemic hemorrhagic disease virus EHDV, and react at 37°C for 1 hour. Serum-free basal medium was added to continue culturing, and the virus was harvested when the cytopathic pathology (CPE) reached more than 75% after 3 to 4 days. The harvested virus liquid was repeatedly frozen and thawed 3 times, centrifuged at 8000 rpm for 30 minutes, the supernatant was taken, and the virus was inactivated by adding formaldehyde with a final concentration of 1 / 3000. Ultracentrifuge at 28,000 rpm and 4°C for 3 hours. The precipitate was dissolved with 1 / 100 of the original volume of PBS (...

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Abstract

The invention relates to a deer epidemic hemorrhage competitive enzyme-linked immunosorbent assay kit and a preparation method and a user thereof, the kit comprises an EHDV antigen-coated ELISA plate,a monoclonal antibody IgG-HRP enzyme conjugate, positive serum, negative serum, 20 times of concentrated washing liquid, 10 times of concentrated dilution buffer, a substrate I, a substrate II, a substrate III and stop solution. The preparation method comprises the following steps: a, preparing a deer epidemic hemorrhage virus ELISA-coated antigen, and detecting the safety of the deer epidemic hemorrhage virus ELISA-coated antigen; b, preparing a monoclonal antibody of deer epidemic hemorrhage virus and carrying out preparation and identification on the antibody-horseradish peroxidase (HRP) enzyme conjugate; c, preparing the positive serum and the negative serum; d, preparing the deer epidemic hemorrhage virus antigen-coated ELISA plate; e, preparing the 20 times of the concentrated washing liquid and the 10 times of the concentrated dilution buffer, and preparing the substrate I, the substrate II, the substrate III and the stop solution; and f, assembling the kit. The kit is used fordiagnosis, quarantine, detection and epidemiological investigation of deer epidemic hemorrhage and is characterized by strong specificity, high sensitivity and the like.

Description

【Technical field】 [0001] The invention relates to the field of biotechnology, in particular to a detection kit for deer epidemic hemorrhagic disease virus antibody competition enzyme-linked immunosorbent assay and its preparation method and application. 【Background technique】 [0002] Epizootic haemorrhagic disease of deer (EHD) is a seasonal, non-contact viral infectious disease. Deer epidemic hemorrhagic disease virus (EHDV), a member of the genus Orbivirus in the Reoviridae family, is a double-stranded RNA virus transmitted by arthropods (mainly Culicoides). Bluetongue virus (BTV) is very similar, and serologically, it has obvious crossover with bluetongue disease. Under natural conditions, deer epidemic hemorrhagic disease virus can cause cattle, sheep and other domestic animals, as well as white-tailed deer (Odocoileus virginianus), millet, eland antelope and other domesticated and wild ruminants infection. The virus has been isolated in the United States from white-t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/535
Inventor 花群义阮周曦杨俊兴杨云庆曾少灵林庆燕吕建强陈兵秦智锋陶虹
Owner 花群义
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