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Method for detecting peste des petits ruminants virus antibody by double-antibody blocking ELISA (enzyme-linked immunosorbent assay) method

A PPR, double-antibody technology, applied in the fields of resisting vector-borne diseases, measuring devices, biological tests, etc., can solve the problems of insufficient fast and accurate detection of PPR antibody detection products, and achieve the improvement of the level of disease prevention and control technology , easy to promote the application, the effect of accurate promotion of the application

Pending Publication Date: 2022-08-09
天津市动物疫病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of the prior art, the present invention provides a double-antibody blocking ELISA method for detecting antibodies against Peste des petits ruminants virus, which solves the problem that the detection products of Peste des petits ruminants antibodies in the prior art are not fast enough and accurate enough

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  • Method for detecting peste des petits ruminants virus antibody by double-antibody blocking ELISA (enzyme-linked immunosorbent assay) method
  • Method for detecting peste des petits ruminants virus antibody by double-antibody blocking ELISA (enzyme-linked immunosorbent assay) method
  • Method for detecting peste des petits ruminants virus antibody by double-antibody blocking ELISA (enzyme-linked immunosorbent assay) method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Preparation of Peste des petits ruminants virus N protein antigen

[0016] 1 Experimental material

[0017] Escherichia coli DH5a, BL21 strains, and Marker were purchased from Beijing Transgen Biotechnology Co., Ltd.; restriction enzymes EcoR I and Xho I were purchased from Dalian Bao Bioengineering Co., Ltd.; protein purification column Ni Sepharose FF was purchased from GE Healthcare Life Science Company; serum Samples are kept for this laboratory.

[0018] 2Transformation, expression and identification of protein expression plasmids

[0019] According to protein sequence https: / / www.ncbi.nlm.nih.gov / gene / 3119775

[0020] Synthetic template, total length is 1575bp (SEQ ID NO.1), according to molecular biology method, use PCR to amplify N gene fragment, use restriction endonuclease EcoR I and Xho I to clone this fragment on prokaryotic expression vector pET28a, pass The plasmid was extracted after restriction enzyme digestion and nucleotide sequence iden...

Embodiment 2

[0041] Example 2 Preparation, purification and horseradish peroxidase labeling of rabbit polyclonal antibody against N protein

[0042] The purified N protein was used to immunize New Zealand white rabbits. After 6 immunizations, the serum was collected and the titer was >10^5 determined by ELISA. Finally, all serum was collected, and the antibody was purified by saturated ammonium sulfate precipitation and analyzed by SDS-PAGE. Then carry out horseradish peroxidase labeling according to the sodium periodate method described by Guo Chunxiang et al. [6] , add 100 μl calf serum and 500 μl glycerol, and store at -20 degrees after mixing.

Embodiment 3

[0043] Embodiment 3 Blocking method ELISA coating antigen optimal concentration and enzyme conjugate antibody optimal dilution selection

[0044] Determine the optimal concentration and optimal dilution according to the square matrix method. Using N protein as the coating antigen, N protein solutions with final concentrations of 1.0, 3.0, 6.0 and 9.0 μg / ml were prepared respectively, and the HRP-labeled antibodies were diluted to 1: 400, 1: 800: 1: 1600: 1: 3200, coat 96 microwell plates with prepared N protein solution, overnight at 4°C, block with 1% BSA in PBS (pH 7.2) for 3h, wash with PBST After 3 times, 50 μl of the above diluted enzyme-labeled antibodies were added, and 50 μl of negative serum samples were added to the corresponding wells, and the samples were repeated 3 times. Warm bath at 37°C for 1 h, shake off the liquid in the wells, wash 5 times with PBST, add color developing solution and keep in the dark for 15 min, add stop solution, measure the OD value of eac...

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Abstract

The invention discloses a method for detecting a peste des petits ruminant virus antibody by a double-antibody blocking ELISA (Enzyme-Linked Immunosorbent Assay) method. The method comprises the following steps: cloning and expressing N protein, immunizing a rabbit with the purified N protein to prepare a polyclonal antibody of the N protein, collecting the polyclonal antibody by adopting a saturated ammonium sulfate precipitation method, and labeling the polyclonal antibody with horse radish peroxidase. A double-antibody blocking ELISA detection method for detecting the peste des petits ruminants virus (PPRV) is established by using a purified N protein coated 96-well plate and an enzyme conjugate polyclonal antibody as a detection antibody, 200 serum samples are detected by using the method determined by the research, the coincidence rate is 95%, and the method can be used for detecting the antibody level of the PPRV in the serum, and can be used for detecting the antibody level of the PPRV in the serum. And a basis is provided for prevention and control of peste des petits ruminants and detection of vaccine effects in the future.

Description

technical field [0001] The present invention relates to a double antibody blocking ELISA method for detecting PPR virus antibodies. Background technique [0002] Peste des petits ruminants (PPR) is an acute infectious disease caused by peste des petits ruminants virus (PPRV). The morbillivirus genus in the family paramyxoviridae is indeterminate in shape, but in most cases it is spherical. Compared with rinderpest virus particles, the particles are generally larger in size, and it is a A single-stranded negative-stranded RNA virus, the nucleocapsid is composed of N protein, which is relatively conservative and has strong immunogenicity. It is mainly prevalent in small ruminants, including sheep, goats, etc. The main feature of the disease is fever , pneumonia, diarrhea and stomatitis, the disease is mainly prevalent in central and western Africa and parts of Asia, including India, Nepal, Pakistan and other countries [1-3] , while the Peste des petits ruminants were first d...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/68G01N33/58G01N33/535
CPCG01N33/56983G01N33/6854G01N33/581G01N33/535G01N2333/115G01N2469/20Y02A50/30
Inventor 袁雪涛石瑜姜德州王芳蕊杨颖杨爱华杨丹张双喜刘伟张鑫
Owner 天津市动物疫病预防控制中心
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