Development Of Indirect Enzyme-linked Immunosorbent Assay And Preparation Of Monoclonal Antibodies Of Campylobacter Fetus Surface Layer Protein SapA

Two gene fragments SapA-N (1398bp) and SapA-C (1422bp) from campylobacter fetus (C. fetus) Surface layer protein gene (SapA) were amplified using PCR method, and recombinant proteins rSapA-N and rSapA-C in the Ecoli BL21 were expressed.Western blot analysis showed that the immunological activity of the rSapA-N was higher than that of the rSapA-C (P > 0.05).The indirect ELISA method was established using rSapA-N. The specificity and sensitivity of the method were proved to be 94.3% and 88.6% respectively. The indirect ELISA method was used to detecte sera samples of abortion cows.The results showed that there were 4 C. fetus positive sera samples in 70 brucellosis positive sera samples, 2 C. fetus positive sera samples in 32 infectious bovine rhinotracheitis positive sera samples and 7 C. fetus positive sera samples in 24 neosporosis positive sera samples respectively indicating that there was mixed infecting of microorganism in the cow abortion cases.To produce the monoclonal antibody (McAb) against surface layer protein rSapA-N of Campylobacter fetus, Balb/c mice were immunized with the recombinant protein SapA-N. Two hybridoma clones(A2D5, C1B6) secreting antibody against rSapA-N were obtained. Both monoclonal antibodies were proved to belong to IgG1 subclass withκchain. Western blot analysis showed that both monoclonal antibodies reacted with recombinant rSapA-N protein only. The titers of antibody in ascites of BALB/C mice were 1:128 000 (A2D5)and 1:64 000(C1B6) respectively. The concentration of purificated McAb proteins was 4.35mg/mL (A2D5) and 1.03mg/mL (C1B6) respectively.