ELISA detection of B cell epitope mapping of SEB and anti-immunodominant peptide sera reactions with SEB.

<p>(A) B cell epitope mapping of SEB using an overlapping 18-mer peptide ELISA. To determine the immunodominant peptides of SEB, microtiter plates were coated with synthetic overlapping peptides that spanned the entire length of the SEB of MRSA252 or BSA and OVA_192-201 (negative control peptides). Then, antiserum samples from BALB/c mice that were immunized with rSEB plus AlPO₄ adjuvant were detected. The absorbance was read at 450 nm. The absorbances at 450 nm for the peptides SEB_205-222, SEB_97–114 and SEB_247-261 were significantly higher than BSA (P<0.01) and higher than OVA_192-201 (P<0.01). (B) Immunodominant peptide antiserum reaction with SEB using an ELISA. To confirm the reactivity of immunodominant peptide-specific antisera with SEB, BALB/c mice were immunized with KLH-conjugated immunodominant peptides and acquired anti-SEB_205-222, SEB_97–114 and SEB_247-261, respectively. Microtiter plates were coated with native SEB. Then, all of the immunodominant peptide-specific antisera at the dilutions of 1:1000, 1:2000 and 1:4000 were detected using an ELISA. To statistically analyze the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090445#pone-0090445-g002" target="_blank">Figure 2B</a>, we used an unpaired Student's t-test in the software SPSS 13.0. Each bar represents the means ± s.d. of triplicate measurements, and the data shown are representative of two or more independent experiments. At different dilutions, all of the peptide-specific antisera were stronger to bind SEB than normal sera of BALB/c mice (* P<0.05), particularly at the dilutions of 1:1000 and 1:2000 (P<0.01). When the dilutions of the peptide-specific antisera were expanded to 1:4000, the differences were not obvious.