Blocking ELISA kit for detecting bovine parainfluenza virus type 3 antibody and application thereof

A bovine parainfluenza virus and kit technology, which are applied in measurement devices, immunoassays, instruments, etc., to achieve the effects of being simple, fast, technically difficult, and easy to operate.

Pending Publication Date: 2020-09-11
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no blocking ELISA detection method for bovine parainfluenza virus type 3 (BPIV3) antibodies at home and abroad. The present invention aims to establish a blocking ELISA detection kit for detecting BPIV3 antibodies, laying the foundation for clinical detection and related research.

Method used

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  • Blocking ELISA kit for detecting bovine parainfluenza virus type 3 antibody and application thereof
  • Blocking ELISA kit for detecting bovine parainfluenza virus type 3 antibody and application thereof
  • Blocking ELISA kit for detecting bovine parainfluenza virus type 3 antibody and application thereof

Examples

Experimental program
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Embodiment 1

[0039] The establishment of embodiment 1 monoclonal antibody hybridoma cell line

[0040] 1. Antigen preparation: Goat parainfluenza virus type 3 (CPIV3) JS2013 strain (the preservation number of this strain is: CCTCC NO: V201832. The preservation date is: June 26, 2018, and the preservation address is: China, Wuhan, Wuhan University, China Center for Typical Culture Collection, which is disclosed in the patent CN2018109262262) to infect MDBK cells, harvest the virus after 4 days of inoculation, and centrifuge at 10,000rpm for 30min to remove cell debris; the supernatant is ultracentrifuged at 40,000×g for 2h, The supernatant was discarded, and the precipitate was dissolved overnight with an appropriate amount of PBS.

[0041] 2. Immunization of Balb / c mice: immunize 6-8 week-old female BALB / c mice (purchased from the Comparative Medicine Center of Yangzhou University) by subcutaneous multi-point injection of super-isolated virus, and immunize 3 times in total, with an interva...

Embodiment 2

[0047] Example 2 Purification and peroxidase labeling of monoclonal antibodies

[0048] 1. Monoclonal antibody purification: Centrifuge ascitic fluid at 4000rpm at room temperature for 15min, and take the supernatant. Slowly add saturated ammonium sulfate solution drop by drop to the supernatant of ascitic fluid with stirring at 4°C until the saturation of ammonium sulfate in the system reaches 50% to precipitate immunoglobulins, continue stirring for 30 min, and then centrifuge at 13 000 rpm at 4°C For 30 minutes, discard the supernatant, take the precipitate and dissolve it in PBS buffer (0.01M, pH7.4), slowly add saturated ammonium sulfate solution drop by drop under stirring at 4°C until the saturation of ammonium sulfate in the system is 33%. Concentration ammonium sulfate graded immunoglobulin precipitation, continued stirring for 30 min, centrifuged at 13 000 rpm, 4 °C for 30 min, discarded the supernatant, took the precipitate and dissolved it in PBS buffer (0.01M, pH7...

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Abstract

The invention discloses a blocking ELISA kit for detecting a bovine parainfluenza virus type 3 antibody. The kit comprises a pre-coated elisa plate and an enzyme-labeled antibody, the pre-coated elisaplate is an elisa plate coated with purified goat parainfluenza virus type 3; the enzyme labeled antibody is a monoclonal antibody 2E6 labeled by horse radish peroxidase; the monoclonal antibody 2E6is secreted by a hybridoma cell strain 2E6; the hybridoma cell strain 2E6 is preserved in China Center for Type Culture collecting on February 1, 2016, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: C201627, and the hybridoma cell strain 2E6 is classified and named as hybridoma cell strain 2E6. The kit disclosed by the invention is good in specificity, high in sensitivity, good in stability, capable of rapidly, simply, conveniently and efficiently detecting the bovine parainfluenza virus type 3 antibody, low in cost and suitable for high-throughput detection of samples.

Description

technical field [0001] The invention relates to the field of biological detection, and relates to a blocking ELISA kit for detecting bovine parainfluenza virus type 3 antibody and application thereof. Background technique [0002] Bovine parainfluenza virus type 3 (BPIV3) belongs to the Respirovirus genus of the Paramyxoviridae family, and is a single-stranded negative-sense RNA virus with an envelope. In addition to BPIV3, the virus members of this genus also include human parainfluenza virus type 1 and type 3 (HPIV1, HPIV3), goat parainfluenza virus type 3 (CPIV3), ovine parainfluenza virus type 3 (OPIV3), etc. important pathogens of respiratory diseases. BPIV3 is the main pathogen of bovine respiratory syndrome. After infection, it often causes acute clinical symptoms, such as elevated body temperature, loss of appetite, listlessness, runny nose, cough, dyspnea, etc., which are extremely harmful to fattening cattle. At present, BPIV3 is popular in countries all over the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/535
CPCG01N33/577G01N33/56983G01N33/535G01N2469/20
Inventor 毛立李文良李基棕刘茂军杨蕾蕾张纹纹孙敏
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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