Goose parvovirus VP3 protein antigen ELISA detection kit, and detection method and application thereof

A technology of goose parvovirus and detection kit, which is applied in the direction of biological testing, material inspection products, etc., can solve the problems of difficult popularization, cumbersome operation, and inconvenient use, and achieve the effect of improving specificity and overcoming low specificity

Inactive Publication Date: 2019-08-27
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

Benefits of technology

This patented technology uses specialized plates made from viruses that infect ruminants or other animals without causing disease. These plasma cells are lysate extracted from these materials and react chemically with certain substances called histones when exposed to UVA radiation. When this reaction occurs, it produces small amounts of DNA fragments known as papillomavirions - tiny crystals containing different proteins found inside them. By comparing two samples taken together, we get another way to determine if there was any cowpox agent being introduced into their bodies during pregnancy.

Problems solved by technology

This patents describes various techniques used during research on viruses like geese or swallowplume that cause illness similar to chickens' death syndrome called Chikungunya fever (CFT). However, current diagnoses involve testing blood samples from affected birds with specific serum levels obtained through traditional tests involving different steps. These procedures require trained staff and take too long. There also exist concerns about their effectiveness due to factors related to human error.

Method used

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  • Goose parvovirus VP3 protein antigen ELISA detection kit, and detection method and application thereof
  • Goose parvovirus VP3 protein antigen ELISA detection kit, and detection method and application thereof
  • Goose parvovirus VP3 protein antigen ELISA detection kit, and detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Massive expression of VP3 protein

[0045] 1. The recombinant baculovirus GPVP3 / Bac strain was inoculated into insect cells Sf9, cultured at 27° C. for 4 days, the culture was collected, and the supernatant was collected by centrifugation to obtain the F2 generation recombinant baculovirus.

[0046] 2. Inoculate the above-mentioned F2 generation recombinant baculovirus into insect cell Sf9 at an inoculum amount of MOI=5-10, culture at 27° C. for 4 days, collect the culture, centrifuge to obtain the supernatant to obtain the recombinant VP3 protein.

[0047] 3. Inoculate the correctly identified recombinant virus into Sf9 cells with an inoculation amount of MOI=1-10 for mass culture, and collect the culture supernatant by centrifugation to obtain a large amount of recombinant VP3 protein.

Embodiment 2

[0048] Example 2 Purification of VP3 protein

[0049] Get the supernatant of the non-inactivated recombinant GPV VP3 protein expressed in the above-mentioned Example 1 and centrifuge at 12000rpm / min for 30min, remove the cell debris, take the supernatant and put it into a dialysis bag (molecular weight 8000-12000), and use poly Ethylene glycol 6000 embedding concentrated 4 times, concentrated and transferred to a 30ml centrifuge tube, 30000rpm / min. Centrifuge at 4°C for 4 hours, discard the supernatant, suspend the pellet at the bottom of the tube with a small amount of PBS, dissolve overnight at 4°C, add cesium chloride to dissolve evenly, and centrifuge at 40,000 rpm / min at 10°C for 24 hours. Use a syringe to suck out the visible bands respectively, determine the target bands by SDS-PAGE electrophoresis and identify the purity, use BCA to quantify the protein content, and scan the electron microscope for analysis, see figure 1 . Aliquot a small amount and store at -70°C. ...

Embodiment 3

[0050] The preparation of embodiment 3 ELISA kit

[0051] The preparation process of the ELISA kit of the present invention is shown in figure 2 , the specific preparation method comprises the following steps:

[0052] A. the preparation method of the polyclonal antibody of described anti-goose parvovirus VP3 protein, comprises the following steps:

[0053] (1) Routinely immunize rabbits with the purified GPV VP3 protein. When the ELISA titer of rabbit serum reaches above 1:10000, collect rabbit blood and centrifuge to obtain serum.

[0054] (2) Purifying the rabbit serum prepared in step (1) through a Protein A column to obtain a purified polyclonal antibody against GPV VP3 protein.

[0055] (3) Add 50% neutral glycerol to the polyclonal antibody against GPV VP3 protein prepared in step (2), measure the working concentration, and store it at -20°C for later use.

[0056] B. The steps of screening positive hybridoma cells by ELISA method:

[0057] (1) Determine the protei...

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Abstract

The present invention discloses a goose parvovirus VP3 protein antigen ELISA detection kit and a detection method thereof. The kit comprises: an ELISA plate for a polyclonal antibody coated with gooseparvovirus VP3 protein, confining liquid, a sample diluent, a purified goose parvovirus VP3 recombinant protein antigen standard substance, an HRP-labeled anti-goose parvovirus VP3 monoclonal antibody, a washing solution, an enzyme substrate A solution, an enzyme substrate B solution, a stop buffer, and an antigen standard substance. The present invention also discloses a method for accurately and quantitatively detecting the goose parvovirus VP3 protein antigen in the sample by using the kit. In addition to the advantages of the ELISA kit, by using the goose parvovirus VP3 protein antigen ELISA detection kit provided by the present invention, the defects such as the low specificity caused by the antigen spatial conformation, the sensitivity caused by the secondary antibody, and the likeare further overcome, and the specificity and sensitivity of the detection are significantly improved.

Description

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Claims

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Application Information

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Owner 扬州优邦生物药品有限公司
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