Recombinant rP1-I, rP1-II, rP1-III and rP1-IV proteins are immunogenic High antibody responses were seen against
each of the four recombinant proteins. The time course response for each of the recombinant proteins showed that the antibody titers gradually increased Selleckchem PLX3397 after first and second booster and peaked after the second boost. An additional figure file [see Additional file 1] shows the time dependent response for recombinant P1-I protein. Almost similar antigenic responses were observed for other three P1 protein fragments (data not shown). The end point titers for each protein were > 1 × 105. Western blotting for all the four recombinant proteins with their respective antibodies confirmed the specificity of each antibody. All these antibodies showed major reactivity with ~170 kDa band of P1 protein in M. pneumoniae lysate by ELISA (Figure 3B) and western blotting. Anti-P1 antibodies also reacted with few additional bands in M. pneumoniae lysate. These additional bands probably represent the degraded P1 protein bands (Figure 3A). No cross reactivity was observed between each of the four antibodies (Figure 3C & 3D). Almost
similar reactivity was observed with two other P1 protein fragments rP1-II & rP1-III (data not shown). These results indicated that all the four P1 protein fragments P005091 are immunogenic and antibodies are specific as they only recognized the corresponding protein fragment. Pre-bleed and control Selleckchem CAL-101 rabbit sera showed no reactivity with any of the recombinant protein fragments. An additional figure
file [see Additional file 2] shows the reactivity of each protein fragment with pre-bleed sera. Figure 3 Western blot and ELISA analysis of M. pneumoniae lysate and Cross reactivity of Pab (rP1-I) and Pab (rP1-IV). Reactivity of P1 (170 kDa) with anti-P1 protein fragments antibody Pab (rP1-I), Pab (rP1-II), Pab (rP1-III) & Pab (rP1-IV) rose in Rabbit by western blotting (A) and by ELISA (B). (C) &(D) Immuno blot analysis of rP1-I, rP1-II, rP1-III and rP1-IV fragments with Pab (rP1-I) and Pab (rP1-IV) showing their cross reactivity with respective sera. Lane Marker: L-NAME HCl Molecular mass marker (kDa). Recombinant rP1-I, rP1-II, rP1-III and rP1-IV proteins were recognized by anti-M. pneumoniae antibody and by sera of M. pneumoniae infected patients All the four recombinant proteins were analyzed for their reactivity to anti-M. pneumoniae antibody and pooled sera of M. pneumoniae infected patients. To do so, 1 μg of each recombinant protein was loaded on SDS-PAGE gel (Figure 4A-I) and the proteins were blotted to nitrocellulose membrane. As shown in Figures 4A-II & III, all the four proteins showed similar reactivity with either of the two sera. We next compared the reactivity of the four recombinant proteins with fifteen and twenty-five sera of M. pneumoniae infected patients by western blot analysis and by ELISA respectively. Figures 4B & 5A shows the reactivity of the recombinant proteins with sera of M.