Identification Of The Binding Peptides Of One Monoclonal Antibody Against Non-structural Protein3A Of Foot-and-mouth Disease Virus And Development Of A Blocking ELISA Utilizing The3A MAb

Foot-and-mouth disease is a highly contagious and acute disease of cloven-hoofedanimals. Many countries attaches great importance to FMD because it can severelyconstrain international trade of animals and animal products. In China, there is acomplex policy to control the FMD mainly by the preventive vaccination. Thus,diagnostic methods play an important role for FMD control.In order to identify the peptide region covering epitopes recognized by amonoclonal antibody3A24#against non-structural protein3A from foot-and-mouthdisease virus, the protein3A was prokaryotically expressed in two peptides,3A1and3A2, covering1~89and77~153amino acid. Primers targeting the gene3a1and3a2were designed and synthesized, respectively. Then the genes3a1and3a2wereamplified by PCR and cloned into plasmid pET-30a(+)-PES-SUMO. The recombinantplasmids were transformed into E.coli BL21(DE3)pLys and induced for expression byIPTG. The expressed peptides were examined by western blot using monoclonalantibody3A24#to identify the epitope covering peptide. SDS-PAGE analysis showedthat both3A1and3A2peptides were successfully expressed with the respectivemolecular weight as26ku and25ku. Western blot analysis showed that, both3A1and3A2can be recognized by FMDV infected cattle serum，but only3A2can reactwith monoclonal antibody3A24#. The epitopes recognized by monoclonal antibody3A24#are located on peptide3A2, namely between the amino acid89~153of FMDVnon-structural protein3A.Differentiating animals infected with foot-and-mouth disease virus (FMDV) fromvaccinated animals has been an important part for controlling FMD. In this research, apolyclonal antibody against NSP2C was produced in rabbit vaccinated withpreviously expressed2C epitope regions. Then a blocking ELISA was developed usedthe polyclonal antibody2C, recombinant protein2C3AB expressed in bacteria and amonoclonal antibody (McAb) recognized an epitope region spanning89~153aminoacids of FMDV NSP3A to detect NSP antibody. The blocking results of cattle andswine serum samples were statistically analyzed, and the critical value was determined that the samples presenting a percentage inhibition (PI) of≥46%wereconsidered positive; samples with a calculated percentage inhibition of <46%wererated negative. This blocking ELISA method proved to be specific, sensitive and itshowed high coincident rate compared with PrioCHECK FMDV-NS ELISA Kitand FMD3ABC I-ELISA Kit. This method will be useful in clinical detection andepidemiological study on FMD for differentiating infection from vaccination.