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ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for goose astrovirus Capsid protein antigen, detection method and application

A detection kit and astrovirus technology are applied in the field of goose astrovirus Capsid protein antigen ELISA detection kits, which can solve the problems of difficult popularization, cumbersome operation, long time consumption, etc., and achieve the effects of fast detection speed and high sensitivity

Pending Publication Date: 2020-06-16
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the disease is prevalent to varying degrees every year, and it is still one of the most important infectious diseases in the goose industry, causing serious losses to the waterfowl industry;
[0003] Although there are many detection methods at present, such as agar diffusion test and agar diffusion inhibition test, neutralization test and animal protection test, direct immunofluorescence diagnosis method, immunoenzyme spot method, but only agar diffusion test method is generally used. There are defects such as cumbersome operation, inconvenient use, long time-consuming and difficult to popularize. Chinese invention patent CN109456391B discloses a VLP vaccine for preventing goose Astrovirus infection and its preparation method and application. The invention adopts the construction of recombinant baculovirus rBac- GoAsV-Capsid expresses the Capsid protein, and a large amount of the Capsid protein is expressed to make a vaccine. Therefore, it is urgent to study an efficient, accurate and quantitative method for the detection of goose Astrovirus

Method used

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  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for goose astrovirus Capsid protein antigen, detection method and application
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for goose astrovirus Capsid protein antigen, detection method and application
  • ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for goose astrovirus Capsid protein antigen, detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Massive expression of Capsid protein

[0034]Step S1: Inoculate the recombinant baculovirus rBacmid-GoAsV-capsid strain into insect cells Sf9, culture at 27°C for 4 days, collect the culture, centrifuge and take the supernatant to obtain the F2 generation recombinant baculovirus ;

[0035] Step S2: Inoculate the F2 generation recombinant baculovirus obtained in step S1 into insect cell Sf9 at an inoculum amount of MOI of 10, culture at 27°C for 4 days, collect the culture, and centrifuge to take the upper Clear the recombinant Capsid protein;

[0036] Step S3: Identify the recombinant Capsid protein prepared in step S2, inoculate the correctly identified recombinant virus with an MOI value of 5 to inoculate Sf9 cells for mass culture, and collect the culture supernatant by centrifugation to obtain a large amount of recombinant Capsid. protein.

[0037] The recombinant Capsid protein is a recombinant baculovirus rBacmid-GoAsV-capsid strain developed by Yangzhou Youban...

Embodiment 2

[0039] Purification of Capsid protein

[0040] Step S1: Centrifuge the supernatant of the culture medium obtained in Example 1 at a rotational speed of 12,000 rpm / min for 30 minutes, remove cell debris, obtain a supernatant with a molecular weight of the recombinant Capsid protein of 10,000, and put the supernatant into Put it into a dialysis bag, and use polyethylene glycol 6000 to embed and concentrate 4 times at a temperature of 4°C to obtain a Capsid protein concentrate;

[0041] Step S2: Add the Capsid protein concentrate prepared in step S1 into a 30ml centrifuge tube, centrifuge for 4 hours at a speed of 30,000rpm / min and a temperature of 4°C, discard the supernatant, and add PBS to the centrifuge tube until Suspend the precipitate at the bottom of the tube, dissolve it overnight at 4°C, add cesium chloride until the precipitate dissolves evenly, and centrifuge for 24 hours at a speed of 40,000rpm / min and a temperature of 10°C to obtain a centrifuge After the solution;...

Embodiment 3

[0044] Preparation of goose astrovirus positive allantoic fluid

[0045] Step S1: Under aseptic conditions, the kidney tissue of the affected goose was taken as the material for virus isolation, and the tissue was added to sterilized saline at a ratio of 1:4 and ground for treatment, repeated freezing and thawing three times, and using 8000×g Centrifugal force, after centrifuging for 10 minutes, take the supernatant, filter and sterilize it through a 0.22 μm sterile filter, and store the filtered supernatant at -20°C for later use;

[0046] Step S2: Aseptically inoculate the filtered supernatant obtained in step S1 onto 10-day-old goose embryos via the chorioallantoic cavity route, each inoculated with 0.2 mL, and set a negative control with sterile saline, at a temperature of Incubate at 37°C, light the embryos once a day in the morning and evening, discard the goose embryos that died within 24 hours, observe continuously for 5 days and record the death of goose embryos and c...

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Abstract

The invention discloses an ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit for a goose astrovirus Capsid protein antigen, a detection method and application. The kit comprises: an ELISA platecoated with a goose astrovirus Capsid protein polyclonal antibody, a confining liquid, a sample diluent, a purified goose astrovirus Capsid recombinant protein antigen standard substance, an HRP labeled anti-goose astrovirus Capsid monoclonal antibody, a washing liquid, an enzyme substrate solution A, an enzyme substrate solution B and a stop solution; meanwhile, the invention further discloses amethod for accurately and quantitatively detecting the goose astrovirus Capsid protein antigen in a sample by adopting the kit. The kit contains an antigen standard substance, can detect the accuratecontent of the goose astrovirus Capsid protein in a sample, and is suitable for quantitative detection of the goose astrovirus Capsid gene subunit vaccine.

Description

technical field [0001] The invention belongs to the field of detection kits, and in particular relates to a goose astrovirus Capsid protein antigen ELISA detection kit, a detection method and an application. Background technique [0002] In recent years, goose flocks in Shandong, Anhui, Jiangsu, Liaoning, Henan and Guangdong have broken out an infectious disease characterized by gout. The disease mainly occurs in goslings aged 5-20 days. Severe urate deposition occurs in organs and joint cavities, and the mortality rate can reach up to 50%, which has caused huge economic losses to the goose industry. A large number of samples have been tested and the positive detection rate of avian astrovirus has reached 96.5% (138 / 143 ), the ORF1b gene of the Shandong plain isolate (SDPY strain) was analyzed for genetic evolution. The acid homology is only 49.5%-67.7%. Animal regression test results show that 1-day-old healthy goslings died one after another 24 hours after being inoculate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/543
CPCG01N33/577G01N33/56983G01N33/54306G01N2333/08G01N2469/10
Inventor 阮菁丁国伟范娟王钜华潘杰刘俊斌魏荣荣孙舒桑建君
Owner 扬州优邦生物药品有限公司
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