Preparation Of A Monoclonal Antibody Against 3B Protein Of Foot-and-mouth Disease Virus, Identification Of The Antigenic Epitope And Establishment Of A Competive ELISA Method

To prepare the monoclonal antibodies(McAbs) against 3B protein of Foot-and-Mouth disease virus, full length of gene 3B was amplified by PCR with the sample of FMDV/JS/05, then it was directed cloned into pET-30 a vector to prokaryotic expression and purification. The FMDV/JS/05 and the purified 3B protein were used as the antigen for ELISA test and mice immunization. After 4 times’ immunization, the spleen cells were collected and infused with myeloma cell Sp2/0 follow ratio 5:1. After selection with indirect ELISA and limiting dilution in 4 times, a positive monoclonal cell was chosen to clonal propagation, the cells were collect to prepare ascites, then its subtype, reactogenicity and the titer were tested. Then 3B gene was split and cloned into pGEX-4T1 vector to GST-fusion expression, and those reactogenicity with the 3B antibody were tested by indirect ELISA after purification. The serological positive and negative serum of FMDV were screened by 3ABC indirect ELISA, which were used with the 3B antibody to build a competitive ELISA method for FMDV antibody detection.As a result, one hybridoma cells line against 3B protein of FMDV was obtained, the subtype was IgM, the titer was 1:12800, and it has good reactogenicity with 3B protein of FMDV in Western blotting and indirect immunofluorescence test. SDS-PAGE results show that the GST fusion 3B split protein were successfully expressed and purified, and indirect ELISA tests show that the 3B1 was specifically recognized by 3B antibody. Four serological positive and negative serum of FMDV were screened by 3ABC indirect ELISA, which were used with the 3B antibody to build a competitive ELISA method, and the results show that the competive ELISA method had 100% coincidence rate with 3ABC indirect ELISA method.