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General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit

A duck hepatitis A and detection kit technology, applied in the biological field, can solve the problems of cumbersome operation, double the cost, limited popularization and application, etc., and achieve the effects of high specificity and sensitivity, good antigenicity, and good safety.

Active Publication Date: 2014-10-08
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After the vaccine is successfully developed, how to track and monitor immune antibodies? The traditional serum neutralization test cycle is long, time-consuming and laborious, which is not conducive to the rapid 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. The ELISA method established by the main antigen protein expressed in Escherichia coli has been reported for one of the serotypes of DHAV. When evaluating vaccines, DHAV-1 and DHAV-3 antibodies need to be tested separately, which is cumbersome and costs double
There is no report on the general ELISA detection method for DHAV-1 and DHAV-3 antibodies

Method used

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  • General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit
  • General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit
  • General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit

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Experimental program
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Effect test

preparation example Construction

[0042] 5. Preparation of Positive and Negative Controls

[0043] The positive serum obtained by DHAV-1VP3-3VP1 recombinant protein immunization was diluted 1:200 with sample diluent (OD 650nm ≥1.0), add the penicillin streptomycin of 1000U / mL, filter aseptically, as the positive control in the general-purpose hepatitis A virus antibody indirect ELISA detection kit; Screen the negative serum (OD 650nm ≤0.25), add 1000U / mL of penicillin streptomycin, sterile filter, as a negative control in the general-purpose hepatitis A virus antibody indirect ELISA detection kit.

[0044] 6. Preparation of chromogenic solution

[0045] Chromogenic solution: Weigh 200mg of tetramethylbenzidine (TMB), dissolve it with 100mL of absolute ethanol or DMSO, and dilute to 1000mL with double distilled water; 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 peroxide, distilled water to 1000mL, adjust the pH value to ...

Embodiment

[0058] 1. Universal duck hepatitis A virus antibody ELISA detection kit, including the following components:

[0059] 1) ELISA strips (96 wells): 5 pieces

[0060] 2) 10× concentrated washing solution: 400mL (diluted 1:10 before use)

[0061] 3) Sample diluent: 200mL

[0062] 4) Goat anti-duck enzyme-labeled secondary antibody (enzyme conjugate working solution): 50mL

[0063] 5) Chromogenic solution: 100mL

[0064] 6) Stop solution: 60mL

[0065] 7) Positive serum control (+): 2mL

[0066] 8) Negative serum control (-): 2mL

[0067] 2. Operation steps:

[0068] 1. Dilute the serum to be tested at 1:200 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;

[0069] 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, and pat dry;

[0070] 3. Add 100 μL o...

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Abstract

The invention discloses a general type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit which comprises an ELISA plate coated with DHAV (Duck Hepatitis A Virus)-1VP3-3VP1 recombinant protein. The DHAV-1VP3-3VP1 recombinant protein is obtained through a method which comprises the following steps: respectively amplifying and cloning a DHAV-1VP3 gene and a DHAV-1VP1 gene by taking DHAV-1 and DHAV-3 as materials to obtain recombinant plasmids pMD-1VP3 and pMD-3VP1; then directionally inserting into a pET-32a(+) expression vector in series, and screening to obtain a recombinant expression plasmid pET-1VP3-3VP1; and carrying out ITPG (Isopropyl beta-D-thiogalactoside) inducible expression and purification to obtain the DHAV-1VP3-3VP1 recombinant protein. The general type duck hepatitis A virus antibody ELISA detection kit disclosed by the invention can be used for evaluating the levels of DHAV-1 and DHAV-3 antibodies through primary detection and is low in cost, easy, convenient and fast to operate and suitable for the detection of batch samples.

Description

technical field [0001] The invention relates to a general duck hepatitis A virus antibody detection kit, which is used for rapid detection of duck hepatitis A virus type 1 (DHAV-1) and type 3 (DHAV-3) antibodies, and belongs to the field of biotechnology. Background technique [0002] Duck viral hepatitis is an acute and highly fatal infectious disease that harms ducklings. It has the characteristics of acute onset, short course of disease, rapid transmission, and high mortality. It mainly affects ducklings under 3 weeks old and seriously threatens the healthy development of the duck industry. . The pathogen is duck hepatitis A virus (Duck hepatitis A virus, DHAV), which belongs to the genus Avian Hepadnavirus of the Picornaviridae family. Due to the significant differences between the DHAV sequences and the lack of cross-reactivity between different types of DHAV, the DHAV is divided into DHAV-1, DHAV-2 and DHAV-3, which correspond to genotype A (serum type 1), genotype B ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C07K19/00G01N33/68G01N33/543
Inventor 马秀丽黄兵李玉峰于可响刘存霞宋敏训刘玉山史玉颖
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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