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Sandwich ELISA detection reagent for detecting Peste des petits ruminants virus and its preparation and use method

A technology for PPR and detection reagents, which is applied in the directions of measuring devices, instruments, scientific instruments, etc., to achieve the effects of low detection cost, high detection throughput, and low detection environmental conditions.

Active Publication Date: 2015-09-02
艾军 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Sandwich ELISA detection kit for Peste des petits ruminants virus, there is no related product commercialized in China

Method used

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  • Sandwich ELISA detection reagent for detecting Peste des petits ruminants virus and its preparation and use method
  • Sandwich ELISA detection reagent for detecting Peste des petits ruminants virus and its preparation and use method

Examples

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Effect test

preparation example Construction

[0056] A preparation method of sandwich ELISA detection reagent for detecting Peste des petits ruminant virus. The preparation method of monoclonal antibody to Peste des pete ruminants of the present invention comprises: immunizing BALB / c mice with purified Pete des petits ruminants virus, taking spleen cells of the immune mice, and The spleen cells and SP2 / 0 myeloma cells are fused with a fusion agent to screen and obtain the anti-epidemics ruminant. The screening process is that the spleen cells and SP2 / 0 myeloma cells are fused with a fusion agent and then selected to obtain hybridoma cell line 5B11. The hybridoma cells are taken and injected into the abdominal cavity of mice to prepare ascites. The 5B11 monoclonal antibody purified by a ProteinG column .

[0057] The polyclonal antibody against Peste des petits ruminants of the present invention is prepared by the following method: immunizing 18-month-old goats with a commercialized Pedeste des ruminants vaccine plus equal vo...

Embodiment

[0096] The kit assembled by the present invention includes: Peste des petits ruminant monoclonal antibody, multiple antibodies to Peste des pete ruminants, diluent, coating solution, HRP-labeled goat anti-mouse secondary antibody, positive control of Peste des petits ruminant virus, negative control of Peste des petits ruminant virus , 20 times concentrated washing solution, substrate solution, stop solution, enzyme-linked immunosorbent plate. The diluent formula is PBS containing 0.5% BSA and 0.05% Tween-20; 20-fold concentrated washing solution is 20-fold concentrated 0.01mol / L pH 7.4 PBS containing 0.05% Tween-20; termination solution is 1 mol / LH 2 SO 4 Solution.

[0097] The product prepared according to the content of the technical scheme of the present invention-a sandwich ELISA test kit for detecting Peste des petits ruminants virus, the reference volume or quantity is as follows:

[0098]

specific example

[0100] 1. Preparation of multiple resistances to Peste des ruminants virus:

[0101] The commercial small animal disease vaccine for ruminants was added with equal-volume Freund’s complete adjuvant (FCA) emulsification and Freund’s incomplete adjuvant (FIA) emulsification. The goats were immunized three times at the age of 18 months. The first immunization was a mixture of FCA and virus. 2. The third immunization is a mixture of FIA and virus; on the 7th day after the end of the last immunization, a small amount of blood is taken from the jugular vein, the serum is separated, and the antibody titer in the serum is analyzed by competitive ELISA. If the test is positive, a large amount of the neck Take blood and separate serum.

[0102] Take 20ml of the positive serum of Peste des Ruminants, add 20ml of normal saline, and add (NH 4 ) 2 SO 4 10ml of saturated solution to make 20% (NH 4 ) 2 SO 4 Stir while adding the solution. After mixing well, let it stand for 30min; centrifuge at 30...

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Abstract

The invention relates to a sandwiched ELISA (Enzyme-Linked Immunoassay) detection reagent for detecting peste des petits ruminant viruses as well as a preparation and use methods of the reagent. The sandwiched ELISA detection reagent comprises a peste des petits ruminant multi-antibody and a peste des petits ruminant monoclonal antibody which are respectively arranged. The sandwiched ELISA detection reagent has the beneficial effects that the detection on the peste des petits ruminant viruses in a clinical sample can be rapidly carried out; compared with a conventional universal peste des petits ruminant virus PCR (Polymerase Chain Reaction) nucleic acid detection method, the detection reagent disclosed by the invention is low in detection environment condition requirement, short in detection time, high in detection flux and low in detection expense, and can be widely applied to relevant detection institutions such as various local livestock veterinary stations.

Description

Technical field [0001] The present invention relates to the field of virus detection, and in particular to a kit for detecting Peste des petits ruminants virus and a preparation method thereof. Background technique [0002] Peste des petits ruminants virus (PPRV) has a morbidity and mortality rate of 100% and 90% for sheep and goats, respectively. It causes serious socio-economic problems in developing countries and is recognized by the World Organization for Animal Health (OIE). Listed as an important animal disease. The disease was first reported in Côte d’Ivoire. It now occurs in most African countries south of the Sahara and north of the equator, and almost all countries from the Middle East to Turkey. It is also widely spread in India, South Asia, and West Asia. In July 2007, the disease was introduced to Tibet for the first time. [0003] As for the detection methods of Peste des petits ruminants virus, the methods recommended by the World Organization for Animal Health are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/531
Inventor 艾军杨云庆杨俊兴聂福平叶玲玲周晓黎花群义成亚李应国董俊尹尚莲
Owner 艾军
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