Foot-and-mouth disease hybridoma cell lines, monoclonal antibodies, detection reagents and kits

A technology of hybridoma cell lines and monoclonal antibodies, which is applied in measuring devices, botanical equipment and methods, biochemical equipment and methods, etc., and can solve problems such as virus reactivation, poor detection efficiency, and high risk

Active Publication Date: 2015-09-23
IND TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The disadvantages of the traditional inactivated virus production method are: (1) Since foot-and-mouth disease is a highly contagious disease, it is transmitted through the air, and the preparation of inactivated virus requires the use of viruses. Not only the laboratory must meet the third-level negative pressure of the biosafety level In the laboratory, and the virus may reactivate during the experiment, which is highly dangerous
(2) The inactivated virus cannot produce non-structural proteins that are naturally produced by the host in a live infection state, so the detection efficiency is not good

Method used

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  • Foot-and-mouth disease hybridoma cell lines, monoclonal antibodies, detection reagents and kits
  • Foot-and-mouth disease hybridoma cell lines, monoclonal antibodies, detection reagents and kits
  • Foot-and-mouth disease hybridoma cell lines, monoclonal antibodies, detection reagents and kits

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0052] Experimental Example 1: Materials and Methods

[0053] 1.1: Serum

[0054] To test the sensitivity of the detection, 32 serum samples of FMD-positive pigs were collected from 8-week-old pigs 34 days after the experimental challenge with FMD virus O / TAW / 97 strain.

[0055] In order to compare the sandwich ELISA detection kit provided by the present invention with other three commercially available ELISA detection kits, it was obtained from 32 head without specific pathogen (Specific-pathogen-free; SPF) and experimentally infected with foot-and-mouth disease virus A total of 320 serum samples were collected from pigs with O / TAW / 97 virus strain. The aforementioned 320 serum samples were collected at 0, 2, 4, 6, 8, 10, 14, 21, 28, and 34 days after virus infection. In addition, 30 serum samples collected from pigs 28 days after experimental infection with the FMD O / TAW / 97 strain were also tested.

[0056] To test specificity, 255 serum samples were collected from uninfec...

experiment example 2

[0077] Experimental example 2: Expression of 3ABC recombinant protein in Escherichia coli

[0078] Foot-and-mouth disease virus O / TAW / 993ABC gene sequence is taken from the National Center for Biotechnology Information (NCBI) GenBank database, GenBank accession No.AJ539137, the 5595th to 6119th nucleotide region, and its detailed sequence is shown in SEQ ID As shown in NO: 1, it contains a coding region (Coding region) with a length of 525 nucleotides, so the recombinant protein encoded by it is 175 amino acids long (the sequence is shown in SEQ ID NO: 2). Comparing the genome sequences of serotype O of Pan-Asian FMD strains recently isolated from Asia, Africa and Europe, the gene identity is about 97% to 99%.

[0079] Insert the above DNA fragment amplified by reverse transcription-polymerase chain reaction into pETvector expression vector. The aforementioned expression vector was introduced into Escherichia coli BL21DE3 strain competent cells, and cultured in a Luria-Bertan...

experiment example 3

[0081] Experimental example 3: Mouse immunization and preparation of anti-3ABC monoclonal antibody

[0082] The hybridoma cell line producing anti-3ABC recombinant protein monoclonal antibody was prepared via the following method: 200 micrograms of the 3ABC recombinant protein prepared in Experimental Example 1.5 were used as an antigen to immunize BALB / c mice by subcutaneous injection, and the immune The interval is 4 weeks. Three to four days before cell fusion, the mice were boosted by subcutaneous injection of the same amount of antigen in phosphate buffered saline. The spleens of the immunized BALB / c mice were sacrificed, and the obtained spleen cells were fused with Sp2 / 0 myeloma cells to obtain hybridoma cells. After two weeks, the culture supernatants of hybridoma cells were screened by indirect enzyme-binding immunosorbent assay containing recombinant protein of foot-and-mouth disease virus O / TAW / 97 or O / TAW / 99; The culture supernatant of hybridoma cells was screene...

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Abstract

The invention provides a hybridoma cell line and a monoclonal antibody thereof, an immune detection reagent containing the monoclonal antibody and a kit thereof, and an immune detection method. The hybridoma cell line is prepared by fusing parental cells and myeloma cells, and has the characteristics represented by the cell line with the preservation number of PTA-11304. The parental cell line is prepared by the following steps that: prokaryotic cells are adopted to express non-structural protein coded by a 3ABC gene fragment of foot-and-mouth disease virus, the non-structural protein is adopted as antigen to immunize mice, and spleen cells of the immunized mice are taken out to prepare the parental cell line. The monoclonal antibody produced from the hybridoma cell line can provide specific recognition for the 3ABC peptide of the foot-and-mouth disease virus, and do not generate the non-specific reaction with the swine vesicular disease antiserum.

Description

technical field [0001] The present invention relates to a hybridoma cell producing a monoclonal antibody against foot-and-mouth disease virus, its monoclonal antibody, and immunoassay reagents and kits containing the monoclonal antibody, in particular to a sandwich enzyme-linked immunoassay The hybridoma cell line producing the monoclonal antibody against the non-structural protein of foot-and-mouth disease virus, the monoclonal antibody thereof, and the immunoassay reagent and kit containing the monoclonal antibody for the adsorption test. Background technique [0002] Foot-and-mouth disease (FMD; foot-and-mouth disease) is a highly contagious disease of artiodactyls, mainly infecting economic animals such as cattle, pigs and sheep. FMD is often characterized by fever and epithelial blisters and rots of the mouth, tongue, nostrils, feet and nipples. Foot-and-mouth disease virus is a positive-sense RNA virus belonging to the Aphthovirus genus of the Picornaviridae family. I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/18C07K16/18C12N15/42C07K14/09G01N33/577
Inventor 陈姿菡李璠潘居祥钟明华
Owner IND TECH RES INST
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