Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit

A technology for duck hepatitis virus and detection kit, which is applied in the biological field, can solve the problems of being unsuitable for batch sample detection, difficult to purify DHV, and limited in popularization and application, and achieves the effects of simple operation, low cost and good safety.

Inactive Publication Date: 2011-07-20
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For a long time, the detection of duck hepatitis has relied on traditional virus isolation and neutralization tests. This method is time-consuming and laborious, and is not suitable for the detection of batch samples.
The ELISA method established based on the whole virus is difficult to purify DHV, which limits the popularization and application of this method.
There is no report on the ELISA detection method for the new type of duck hepatitis antibody in Korea

Method used

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  • Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit
  • Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit
  • Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit

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preparation example Construction

[0035] 5. Preparation of Positive and Negative Controls

[0036] The positive serum obtained by immunizing with the Korean new type duck hepatitis VP1 recombinant protein was diluted 1:100 with the sample diluent (OD 450nm ≥1.0), add the penicillin streptomycin of 1000U / mL, filter aseptically, as the positive control in the indirect ELISA detection kit of Korean new type duck hepatitis antibody; The negative serum (OD 450nm ≤0.25), add 1000U / mL penicillin streptomycin, filter aseptically, as the negative control in the indirect ELISA detection kit for Korean new type duck hepatitis antibody.

[0037] 6. Preparation of chromogenic solution

[0038] Chromogenic solution A: weigh 200mg of tetramethylbenzidine (TMB), dissolve it with 100mL of absolute ethanol or DMSO, and dilute to 1000mL with double distilled water; Chromogenic solution B: weigh 21g of citric acid (C 6 h 8 o 7 ·H 2 O), 28.2g anhydrous disodium hydrogen phosphate (Na 2 HPO4), 6.4mL 0.75% urea hydrogen peroxi...

Embodiment

[0052] 1. Korean New Type Duck Hepatitis Antibody ELISA Detection Kit, including the following components:

[0053] 1) ELISA strip (96 wells): 5 pieces

[0054] 2) 10× concentrated washing solution: 400mL (diluted 1:10 before use)

[0055] 3) Sample diluent: 200mL

[0056] 4) Goat anti-duck enzyme-labeled secondary antibody (enzyme conjugate working solution): 50mL

[0057] 5) Chromogenic solution A: 50mL

[0058] 6) Chromogenic solution B: 50mL

[0059] 7) Stop solution: 60mL

[0060] 8) Positive serum control (+): 2mL

[0061] 9) Negative serum control (-): 2mL

[0062] 2. Operation steps:

[0063] 1. Dilute the serum to be tested 1:100 with the sample diluent, add 100 μL / well to the antibody detection plate, set up a negative serum control and a positive serum control, and incubate at 37°C for 45 minutes;

[0064] 2. Discard the liquid in the reaction wells, add 350 μL of washing solution to each well, wash 3 to 5 times with an interval of 1 min between each time, a...

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Abstract

The invention discloses a Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit. The detection kit contains an ELISA board coated by Korean novel duck hepatitis VP1 (Phenotypic Variance1) recombination protein, a sample diluent, concentrated washing liquid, an enzyme conjugate working solution, a chromogenic reagent (A), a chromogenic reagent (B), a stopping solution, a positive contrast solution and a negative contrast solution. The VP1 recombination protein is obtained by using the following method: using Korean novel duck hepatitis viruses as a material, augmenting and cloning the VP1 gene through an RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) method to obtain recombinant expression plasmid pMD (physical medium dependent)-VP1; then, directionally inserting to an expression vector pET-32a (+) and screening to obtain recombinant expression plasmid pET-32a(+)-VP1; and inducing, expressing and purifying by ITPG (Isopropyl beta-D-Thiogalactopyranoside) to obtain VP1 recombination protein. The detection kit is used for detecting the Korean novel duck hepatitis and has strong specificity, high sensitivity, simplicity of operation, easiness of popularization and application in a large-area range and wide market prospects.

Description

technical field [0001] The invention relates to a novel duck hepatitis virus antibody detection kit, which is specially used for the rapid detection of Korean novel (genotype C) duck hepatitis virus antibody, and belongs to the field of biotechnology. Background technique [0002] Duck viral hepatitis is an acute, highly fatal and contact infectious disease that harms ducklings. Duck hepatitis virus is usually divided into three serotypes, namely type 1 (DHV-1), type 2 (DHV-2) and type 3 (DHV-3). Type 1 DHV has a worldwide distribution. In recent years, a new type of duck hepatitis has been reported in many areas of China, which has significant differences in sequence with DHV-1, and none of the new DHVs cross-reacts with DHV-1, which seriously restricts the development of the duck industry. In order to distinguish the above strains, domestic scholars call them "Taiwan new type", "Korean new type" and "serotype 1" DHV respectively. The current study found that DHV such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/51C12P21/02G01N33/576G01N33/569G01N33/535
Inventor 马秀丽黄兵李玉峰吴静于可响宋敏训秦卓明
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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