Development Of A Monoclonal Antibody-based ELISA To Detect Foot-and-Mouth Disease Virus (FMDV) And Fusion Expression Of FMDV VP1-VP3-3C Antigens

Foot and mouth disease virus, FMDV, is the etiology of a highly contagious animal disease which can be pandemic and cause a severe economical loss worldwide. this disease also has an imoprtant political impact on the afftected nations. The infections happened in human were reported. It has been well realized that development of an effective vaccine, alongside with accurate diagnostic methods, plays a key step in the control and prevention of the disease. Currently, many assays for detection of FMDV have been develped, amongst which an inactivated virus-based ELISA is used to detect antibodies against FMDV. As the manipulation of live virus is involved in process of the preparation of viral antigen, it is risky in dissemination of viruses. More importantly, it is a "gold standard" method to detect virus antigen for confirmation of FMDV infection. A monoclonal antibody-based ELISA for detection of FMDV is supposed to meet the requirement of sensitivity and specificty, however, no report can be found at present. In addition, the fusion expression of antigens in E.coli is predicted to improve the immunogenicity of the subunit vaccine against FMDV. This paper describes the monoclonal antibody preparation, development of antigen captured ELISA, and eukaryotical expression of FMDV genes encoding antigens.1 Preparation and application of monoclonal antibody against FMDVBALB/C mice were immunized with purified FMDV, followed by the fusion of spleen cells with myloma cells, SP2/0, and screening by ELISA. As a result, two hybrodoma cell lines, designated as 2G12 and 2G8, which secreted antibodies against FMDV were developed. The derivation of hybridoma from fusion of two cells mentioned above was identified by analysis of the hybridoma chromosomes. The monoclonal antibodies can specifically recognize FMDV in infected cells by immunofluorescence assay. One of the monoclonal antibodies, 2G12, was used in the test. for further investigating the potential of 2G12 in detecting FMDV by immunoassay, IgG was purifed from rabbit anti-FMDV serum and used as capture antibody to coat microtiter plates. The capture ELISA was optimized by checkerboard assay. Ninety nanogram of FMDV can be detected by this method while no cross reactions with hog cholera virus, porcine respiratory and reproduction syndrome virus, pseudorabies virus, porcine parvavirus, Japanese encephalitis virus were observed. The reproducibility of the assay was alsotested. This ELIS A provides a new sensitive and specific tool for detection of FMDV. 2 Fusion expression of recombinant FMDV VP1-VP3-3C protein in E.coliA1564 bp motif, spanning full length of VP1 gene, partial VP3 and full length of 3C protein, was amplied by PCR and subjected to sequence analysis. The amplified product was enzymaticalUy digested and ligated to pGEX-KG vector to produce an expression plasmid, which was transformed to E.coli BL-21 cells. The protein expression was induced by addition of IPTG and the pelleted cells were analysized by SDS-PAGE and Western blot. The results showed that a fusion protein with size of 83 kDa was expressed and could react with swine anti-FMDV serum. This experiment pave a way for further research on diagnosis and subunit vaccine of FMDV.