Development And Preliminaryapplication Of Monoclonalantibody Against Serotype C VP1Foot-and-mouth Disease Virus

The conventional detection methods of Foot and mouth disease（FMD）are RT-PCR, virusisolated, neutralizing test, multiple antibody sandwich ELISA and so on. Although the results ofthese detection methods are reliable, they need carefully manipulate and the technical difficultyis huge. Therefore, it is very important to build a specific, sensitive, rapid and accurate detectionmethod of FMDV. Now monoclonal antibody （McAb） has widely used in the diagnosis andtreatment of many animal diseases because its high specificity, single phase, easy to standard andpossibility of mass process. In this study, the high specificity monoclonal antibody against C serotype ofFMDV was prepared. It could be used in developing of vaccine, and study of etiology and immunology. Themonoclonal antibody was used to build initially antigen sandwich ELISA method, and this methodcould be used in diagnostic and prevention of C serotype of FMDV.In this study, recombinat plasmid pPRO-CVP1was transformed to BL21（DE3） pLysS.IPTG was used to induce expression of the fusion protein. Then the protein was purified withGST affinity chromatography method and identified by SDS-PAGE and Western blot. Thefemale mouse BABL/c were immunized by emulsified serotype C FMDV. After immunized4times and detected the titers, the hybridoma is prepared by the fusion of mouse spleen cell andSp2/0myeloma cell under the effect of PEG1500, and subcloned by limiting dilution. IndirectELISA method with purified type C FMDV VP1protein was established, and used to screenthe monoclonal antibody. The subtype of McAb was identified by immunoglobulin standardsubtype identification kit. The McAb was purified with two methods, and its biologicalcharacteristics were determined by indirect-ELISA, Western blot, indirect immunofluorescenceassay and relative affinity test. To detect the serotype C FMDV, the purified McAb and positiveserum of guinea pigs against serotype C FMDV were used to develop initially antigen sandwichELISA method, and the test conditions were optimized.In this research, the VP1recombinat protein of C serotype FMDV was successfullyexpressed, and the concentration was3285μg/mL after purification. The concentration ofpurified C serotype FMDV was1074μg/mL. Three strains of monoclonal antibody (3B3,5A6,6D5) were obtained and the subtypes were IgG1, IgG2a,IgG3respectively.The numbers ofchromosome in the three strains are all between101and106. The concentration of3B3,5A6,and6D5after purified were2876μg/mL,3059μg/mL,3481μg/mL. After SDS-PAGE, only theheavy chain and light chain were observed with MW of45kDa and25kDa. Determined byindirect-ELISA, the titers of3B3,5A6,6D5in cell culture supernatant were1:640,1:1280,1:1280, while the titers of3B3,5A6,6D5in ascite were1:25600,1:51200,1:51200. After purified,the concentrations of3B3,5A6,6D5were1:12800,1:25600,1:25600. Biologicalcharacteristics show that the three strains were highly specificity. Their relative affinity was 6D5>5A6>3B3. After the antigen sandwich ELISA method was built initially, the optimalworking concentrations of McAb, multiple antibodies and HRP-IgG were1.74μg/mL,1:2000and1:10000. The optimal closed time was1h, while the optimal substrate reaction time was15min. Sensitivity assay show that virus could be detected at least168ng/mL. Specify test showedthat only the results of VP1protein of C serotype FMDV and C serotype FMDV of sucklingmice adapted were positive, while others were negative. This is show that the detection methodwas high specificity.In this study, three strain McAbs against C serotype FMDV were prepared and theirbiological characters were further analyzed. Based on this, the antigen sandwich ELISA methodwas developed initially. And used to detect the serotype C FMDV which will be helpful in thediagnostic of FMDV.