A double-antibody sandwich ELISA detection kit for prawn white spot disease virus

A detection kit and double-antibody sandwich technology, which is applied in the interdisciplinary field of immunology and virology, can solve the problems of quantitative detection, low sensitivity, expensive equipment, etc., and achieve a large detection concentration range, good repeatability, and low detection cost Effect

Active Publication Date: 2014-10-08
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary PCR has high sensitivity, but the operation is complicated, and quantitative detection cannot be performed; colloidal gold immunochromatographic test strips can obtain detection results within a few minutes, but its sensitivity is not high, and quantitative detection cannot be performed; real-time quantitative PCR has high sensitivity, Can be quantitatively detected, but the equipment is expensive and the operation is complicated

Method used

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  • A double-antibody sandwich ELISA detection kit for prawn white spot disease virus
  • A double-antibody sandwich ELISA detection kit for prawn white spot disease virus
  • A double-antibody sandwich ELISA detection kit for prawn white spot disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation of White Spot Disease Virus (WSSV) Double Antibody Sandwich ELISA Detection Kit

[0031] 1. Preparation of capture antibody and its titer determination

[0032] (1) Preparation of capture antibody: Take 0.5 mg of purified white spot disease virus and mix well with the same amount of complete Freund's adjuvant, and inject New Zealand white rabbits subcutaneously at multiple points; Mix with the same amount of Freund's incomplete adjuvant, and inject New Zealand white rabbits subcutaneously at multiple points; then carry out the second and third booster immunization every other week, and inject the purified white spot virus subcutaneously at multiple points. The antiserum was collected 6 days after the third booster immunization, and the rabbit antiserum was purified by caprylic acid-ammonium sulfate method to obtain the capture antibody.

[0033] (2) Titer determination of capture antibody: Add 100 μL of 20 μg / mL white spot disease virus to each w...

Embodiment 2

[0063] Example 2: Application of kits to detect changes in WSSV content in gill tissues of crayfish artificially infected with white spot disease virus

[0064] 1. Virus infection: divide the healthy shrimps that have been temporarily raised for 7 days into an experimental group and a control group, with 200 shrimps in the experimental group and 200 shrimps in the control group. The experimental group was fed with bait mixed with white spot virus, and the control group was fed with bait without virus. Eight live shrimps were randomly selected from the experimental group and the control group every 12 hours;

[0065] 2. Preparation of samples to be tested: Take a small amount of shrimp gill tissue, add 2 mL of 0.01mol / L phosphate buffer (containing 0.5 M EDTA) to 1 g of tissue, grind in an ice bath for 3-5 min, and place at 4 °C for 4 000 r / min for 10 min, and the supernatant was allowed to stand at 4°C for 1 h to be detected; the hemolymph was collected and anticoagulant was a...

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Abstract

The invention relates to a WSSA (white spot syndrome virus disease) double-antibody sandwiched ELISA (enzyme linked immunosorbent assay) detection kit which is used for detecting a monoclonal antibody of WSSA and comprises the monoclonal antibody as well as a WSSA double-antibody sandwiched ELISA detection method. The monoclonal antibody is secreted by the hybridoma with the title of hybridoma WSSV-3B7, the preservation number of CCTCC (China Center for Type Culture Collection) NO: C201398, the preservation department of China Center for Type Culture Collection, and the preservation date of June 24th, 2013. The detection kit comprises a rabbit anti-WSSV multi-antibody as a capture antibody, a mouse anti-WSSV mono-antibody WSSV-3B7 as a detection antibody, as well as an ELISA plate coating the capture antibody, a confining liquid, a scrubbing liquid, a goat anti-mouse IgG (immunoglobulin G) antibody marked by alkaline phosphatase and a substrate developing liquid. The WSSA double-antibody sandwiched ELISA detection kit and the detection method, which are provided by the invention, have the advantages of quantification, large detection concentration range, high flexibility, high specificity, good reproducibility, short required detection time and the like.

Description

technical field [0001] The invention relates to a virus detection kit, in particular to a double-antibody sandwich ELISA kit for detecting prawn white spot disease virus (WSSV), which belongs to the interdisciplinary technical field of immunology and virology. Background technique [0002] Shrimp white spot disease virus (WSSV) is the only member of the genus Whispovirus in the family Nimaviridae. It is an enveloped, rod-shaped double-stranded DNA virus that can widely infect decapod crustaceans such as shrimp and crabs. . WSSV mainly infects subcutaneous tissue, hematopoietic tissue, connective tissue, lymphoid organs, etc., and the nucleus of the infected tissue is enlarged. The typical symptoms of diseased shrimp are white spots of various sizes on the inside of the carapace, and the carapace is easily peeled off. Once the prawns are infected with the disease, the mortality rate can reach 100% within a few days, causing great losses to the prawn farming industry. The di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C12N5/20G01N33/577
Inventor 战文斌唐小千绳秀珍刘鹿山邢婧
Owner OCEAN UNIV OF CHINA
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