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Sandwiched ELISA (Enzyme-Linked Immunoassay) detection reagent for detecting peste des petits ruminant viruses as well as preparation and use methods of reagent

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

Active Publication Date: 2013-09-18
艾军 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Sandwich ELISA detection kit for Peste des petits rumin

Method used

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  • Sandwiched ELISA (Enzyme-Linked Immunoassay) detection reagent for detecting peste des petits ruminant viruses as well as preparation and use methods of reagent
  • Sandwiched ELISA (Enzyme-Linked Immunoassay) detection reagent for detecting peste des petits ruminant viruses as well as preparation and use methods of reagent

Examples

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preparation example Construction

[0056]A method for preparing a sandwich ELISA detection reagent for detecting Peste des petits ruminants virus. The method for preparing the monoclonal antibody against Peste des petits ruminants of the present invention comprises: using purified Peste des petits ruminants virus to immunize BALB / c mice, taking spleen cells from the immunized mice, and The splenocytes and SP2 / 0 myeloma cells are fused under the fusion agent to screen the obtained PPR monoclonal antibody. The screening process is to fuse the splenocytes and SP2 / 0 myeloma cells under the fusion agent to obtain the hybridoma cell line 5B11, take the hybridoma cells, inject them into the peritoneal cavity of mice, prepare ascites, and use ProteinG column to purify the 5B11 monoclonal antibody .

[0057] The polyclonal antibody against Peste des petits ruminants described in the present invention is prepared by the following method: using a commercialized Peste des petits ruminants vaccine emulsified with an equal v...

Embodiment

[0096] The kit assembled in the present invention includes: Peste des petits ruminants monoclonal antibody, Peste des petits ruminants polyclonal antibody, diluent, coating solution, HRP-labeled goat anti-mouse secondary antibody, positive control of Peste des petits ruminants virus, negative control of Peste des pets ruminants virus , 20 times concentrated washing solution, substrate solution, stop solution, ELISA plate. The diluent formula is PBS containing 0.5% BSA and 0.05% Tween-20; the 20-fold concentrated washing solution is 20-fold concentrated PBS containing 0.05% Tween-20 at 0.01mol / L pH7.4; the stop solution is 1mol / L L H 2 SO 4 solution.

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

[0098]

specific example

[0100] 1. Preparation of polyclonal antibodies against Peste des petits ruminants virus:

[0101] The commercial Peste des petits ruminants vaccine was emulsified with an equal volume of Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA), and immunized 18-month-old goats three times. The first immunization was a mixture of FCA and virus, and the second 2. The third immunization is a mixture of FIA and virus; on the 7th day after 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 blood in the neck Blood was drawn and serum was separated.

[0102] Take 20ml of the positive serum of Peste des Petits Ruminants, add 20ml of normal saline, and then add (NH 4 ) 2 SO 4 Saturated solution 10ml, made into 20% (NH 4 ) 2 SO 4 Solution, stir while adding, after fully mixing, let it stand for 30min; ce...

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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 invention relates to the field of virus detection, in particular to a kit for detecting Peste des Petits Ruminants virus and a preparation method thereof. Background technique [0002] The morbidity and mortality of peste des petits ruminants virus (PPRV) to sheep and goats is as high as 100% and 90%, respectively, causing serious social and 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, and now occurs in most African countries south of the Sahara and north of the equator, almost all countries from the Middle East to Turkey, and is also widely spread in India, South Asia, and West Asia. In July 2007, the disease was first introduced into the Tibet region of my country. [0003] The methods recommended by the World Organization for Animal Health for the detection of Peste des petits ruminants virus...

Claims

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Application Information

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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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