ELISA kit for detecting duck plague virus antibody, and antibody detection method thereof

A technology of duck plague virus and kit, which is applied in the field of veterinary medicine, can solve the problems of time-consuming and laborious, the complexity of the purification method, the purity is not ideal, and the detection of unsuitable serum samples, etc., and achieve the effect of good specificity

Active Publication Date: 2012-02-22
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic method is the serum neutralization test, but its sensitivity is not ideal, and it is time-consuming and laborious, so it is not suitable for the detection of large quantities of serum samples
ELISA has the characteristics of strong specificity, high sensitivity, fast accuracy, and easy operation. It is an effective means for early rapid diagnosis of DPV infection and monitoring of immune antibodies; however, the complexity and purity of DPV whole virus purification methods are not ideal. Large-scale application of the ELISA method of coated whole virus (DPV-ELISA)

Method used

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  • ELISA kit for detecting duck plague virus antibody, and antibody detection method thereof
  • ELISA kit for detecting duck plague virus antibody, and antibody detection method thereof
  • ELISA kit for detecting duck plague virus antibody, and antibody detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of antibody capture agent

[0036] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental method that does not indicate specific conditions in the preferred embodiment is usually according to conventional conditions, such as described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al., translated by Huang Peitang, etc., Science Press, 2002) conditions, or as recommended by the manufacturer.

[0037] One material method

[0038] Strains, plasmids and strains

[0039] Plasmid pMD18-T, purchased from Dalian Bao Biological Engineering Co., Ltd.; prokaryotic expression plasmid pET32b(+), product of Novagen; clone host strain E.coli DH5a, expression host strain E.coli Rossetta(DE3) and virulent strain of DPV CHv , provided by the Poultry Disease Research Center of Sichuan Agricultural University.

[0040] 2 Test duck embryos ...

Embodiment 2

[0115] Example 2 ELISA kit for detecting duck plague virus antibody

[0116] Solid phase carrier: The solid phase carrier is used as an adsorbent and container during the ELISA determination process, and does not participate in chemical reactions. There are many materials that can be used as solid-phase carriers in ELISA, such as polystyrene, which must meet the requirements of strong protein adsorption properties. After antibodies or protein antigens are adsorbed on it, they still retain their original immunological activity and can be made into various forms. . There are three main shapes of ELISA carriers: microtiter plates, beads, and small test tubes. The microtiter plate is the most commonly used, and the product dedicated to EILSA is called an ELISA plate. The international standard microtiter plate is a 96-well format of 8×12. In order to facilitate the detection of a small number of specimens, there are 8-well strips or 12-well strips. After being placed in the ho...

Embodiment 3

[0122] Example 3 The method of using ELISA kit to detect duck plague virus antibody

[0123] one Material

[0124] The purified recombinant UL16 protein obtained in the above examples is resistant to DPV (Duck Plague Virus), DHBV (Duck Hepatitis B Virus), DHV (Duck Hepatitis Virus), RA (Rimerella duck), Salmonella (Duck Salmonella), DSHDV (Duck swollen head hemorrhagic disease virus), H5N1 (influenza virus) and E.coli (duck Escherichia coli) positive duck serum and anti-DPV duck negative serum were provided by the Poultry Disease Research Center of Sichuan Agricultural University; sheep anti-duck IgG-HRP ( Horseradish peroxidase-labeled goat anti-duck IgG) and tetramethylbenzidine (TMB) were purchased from KPL Company in the United States. Goat anti-duck IgG-HRP was a freeze-dried agent, packaged as 0.1mg / bottle, when used According to the instructions, it was dissolved into 1ml; bovine serum albumin (BSA) was purchased from Sigma Company of the United States.

[0125]...

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Abstract

The present invention relates to the field of animal medicine, particularly to an ELISA kit for detecting duck plague virus antibody. The ELISA kit comprises a solid-phase support, an antibody capturing agent, enzyme-labelled second antibody, a substrate and a blocking solution. The antibody capturing agent comprises recombinant duck plague virus UL16 protein, and the nucleotide sequence of the recombinant duck plague virus UL16 protein is represented by SEQIDNO:3. The method for detecting the duck plague virus antibody through the ELISA kit comprises the following concrete steps: preparation of the solid phase antigen, combination of the first antibody, combination of the secondary antibody, color reaction, detection and determination. According to the present invention, the kit provided by the present invention has high specificity and high sensitivity; the intra-assay or inter-assay repeated test results of the method show that: the variation coefficients are less than 10%; the positive serum of the DPV attenuated vaccine immunized duck can be detected, wherein the positive serum is diluted by 5120 folds.

Description

Technical field [0001] The invention involves the field of animal medicine, and specially involves the ELISA kit and antibody detection methods for detecting duck plague virus antibodies. Background technique [0002] Duck Plague (DP) is a high degree of death infectious diseases commonly caused by duck, geese, swan and other water birds caused by duck plague virus (DPV) in the herpes virus.The region is distributed, and its prevention and control have directly related to the continuous and stable development of the aquaculture industry.Clinical and laboratory trials have confirmed that DPV weak poison vaccines are effective biological preparations to prevent and control DP. Monitoring of DPV specific antibodies is the key to evaluating the immune effect of DPV weakly toxic vaccine and formulating reasonable immune procedures.At present, the methods used to detect DPV antibodies include Neutralization Test (NT), enzyme-linked Immunosorbent Assay (Elisa), Dot-Elisa determination, ...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N15/34C12N15/70C07K14/01
Inventor 程安春何琴汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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