Preparation And Identification Of Monoclonal Antibodies Against The Common Antigen Of Porcine Mycoplasmas

The 8-week-old female BALB/c mice were injected intraperitoneally with 250μg membrane protein of heat-killed Mycoplasma hyopneumoniae(Mhp) emulsified in Freund's complete adjuvant. Three weeks later, the same antigen was administered intravenously without adjuvant, and three days after final immunization, spleen cells from immunized mice were fused with Sp2/0-Ag-14 myeloma cells. By using indirect ELISA and direct agglutination screening hybrid cells, and using limiting dilution method to subclone positive clones three times, two hybridoma cell lines secreting monoclonal antibodies (MAbs) against Mhp, designed as 3-1G2 and 3-8B2, were established. The tilers of their ascitic fluids were 5 × 1012'and 5× 1011 in indirect ELISA , and 1:320-1:400 in direct agglutination. Both of them were of IgG1 subtype, and their affinity index were 40 μg/ml, and 54μg/mL, respectively. In Dot-ELISA, two MAbs could react with Mhp and M. hyorhinis (Mh), but neither could bind with avian mycoplasmas nor with control serum samples. The results suggested that two MAbs were probably specific to the common antigen of porcine mycoplasmas.In order to define these common antigens of porcine mycoplasmas, firstly, it was evaluated that whole cell proteins of Mhp and Mh had 15-20 polypeptide bands at the range of 14,000-120,000 molecular weight by SDS-PAGE. Some bands with high molecular weight were faint, whereas the mid- and low-molecular-weight polypeptides (80Kd and 30Kd) were prominent in Coomassie-blue-stained gels. The profiles of Mhp 168 strain F332 in range of 31,000-43,000 molecular weight were more than ones of Mhp 168 strain F99, Mhp J strain and Mh. Secondly, in western blotting analysis, it was shown that the main common antigens of porcine mycoplasmas were 80Kd and 30Kd using rabbit hyperimmune sera as detecting reagents. Two MAbs reacted with one of these common antigens in western blotting assay, but did nol bindwith proteins of swine serum. It suggested that two MAbs specifically recognized the common antigen with 30Kd of porcine mycoplasmas. All these results indicated that two MAbs would be very useful as a tool for the antigenic analysis of Mhp, the serological diagnosis of Mhp, surveillance of vaccine products, and also detection of mycoplasma contamination in cell cultures.