Preparing HPV-16L1/-18L1 antigens and their immunoreactivities.

A. HPV-16 L1/HPV-18 L1 recombinant proteins obtained in this study were detected by western blot. In all membranes, line 1: Dock-Tag purified pupae infected with HPV-16 L1, line 2: Dock-Tag purified pupae infected with HPV-18 L1, line 3: non-infection pupae, and line 4: non-recombination viral infection pupae. B. Purified HPV-16 L1/HPV-18 L1 recombinant proteins or Cervarix^® were captured on plates with 25, 10, 5 ng as antigens and direct ELISA was performed. Cervarix^® contains HPV-16/-18 VLP and was used as a positive control. As a primary Ab, antisera of rabbit #1 (black bar) and rabbit #2 (gray bar) were used. C. Direct ELISA results showed HPV type-specific immunoreactivities with relatively high backgrounds by measuring with the international standard serum for HPV-16 and HPV-18. We used a control serum that was negative to HPV Ags by validation using ELISA with Cervarix^® Ag (S2 Fig). ELISA was performed twice independently in triplicate. Asterisk (*) indicates a significant difference from the control serum (p < 0.05). D. MB-ELISA showed higher type-specific immunoreactivity than the direct ELISA. Values are means of triplicate results, and error bars show standard deviations.