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. 2013 Jul 2;8(7):e67881. doi: 10.1371/journal.pone.0067881

Figure 4. Western blot and ELISA analyses of samples from chinchillas immunized with the His-tagged MhaB protein.

Figure 4

Western blot (panels A, B, C): Equivalent protein amounts were resolved by SDS-PAGE, transferred to PVDF and probed with the indicated primary and secondary Abs. Post-boost serum and lavage samples taken on Day 44 of the vaccination experiments (see Fig. 3) were pooled and used as primary Abs at the dilution shown in parentheses. Goat α-rat Abs conjugated to HRP were used as secondary Abs. Panel A: western blot of outer membrane protein preparations from the WT M. catarrhalis strain O35E and the mhaB1mhaB2 mutant O35E.B1B2. Panels B and C: western blot of the purified recombinant proteins GST-tagged MhaB and GST-tagged McaP (used as negative control). Arrows indicate proteins specifically reacting with chinchilla Abs α-MhaB1/MhaB2. MW markers are shown to the left in kDa. ELISA (panel D): Individual serum samples were serially diluted and placed in duplicate wells of plates coated with GST-tagged MhaB. Goat α-rat Abs conjugated to HRP were used as secondary Abs. The results are expressed as the mean (± std deviation) end-point titer of samples from n = 12 animals. Individual titers were determined using pre-immune samples as background. Open bars correspond to pre-boost samples taken on Day 19 of the vaccination experiments while black bars represent post-boost samples collected on Day 44 (see Fig. 3).