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

<p><b>A.</b> 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>B.</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. <b>C.</b> 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 (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171314#pone.0171314.s002" target="_blank">S2 Fig</a></b>). ELISA was performed twice independently in triplicate. Asterisk (*) indicates a significant difference from the control serum (<i>p</i> < 0.05). <b>D.</b> MB-ELISA showed higher type-specific immunoreactivity than the direct ELISA. Values are means of triplicate results, and error bars show standard deviations.</p

List of vaccines and time-course sample collection.

<p>List of vaccines and time-course sample collection.</p

Change in HPV-16/-18 Ab levels after vaccination.

<p><b>A.</b> A schema of the vaccination and blood sampling protocol. HPV vaccination (Gardasil^®) was provided at 0, 2, and 6 months, and blood sampling was conducted at 0 (pre), 6 (1st), and 18 (2nd) months. <b>B and C.</b> The change in serum HPV-16 (left) and HPV-18 (right) Ab concentrations of each subject was observed at the 1st and 2nd time-point. Asterisk (*) represents p < 0.001, one-way ANOVA. <b>D</b> and <b>E</b>. Correlation between anti-HPV-16 Ab (x-axis) and anti-HPV-18 Ab (y-axis) at 1st (6 months, left) and at 2nd (18 months, right) collection time points.</p

Correlation between IC and MB-ELISA.

<p><b>A.</b> Technical details of IC method to visualize L1 Abs. IC values are given as a visual score, determined by the color chart. (C, control line; T, test line). <b>B and C</b>. Scatter plots of IC (x-axis) and MB-ELISA (y-axis) from the same samples for anti-HPV-16 and HPV-18 Ab levels. <b>B.</b> The same samples in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171314#pone.0171314.g002" target="_blank">Fig 2</a></b> were measured by IC. <b>C.</b> The same samples in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171314#pone.0171314.g003" target="_blank">Fig 3</a></b> were measured by IC. Five samples were arbitrarily selected from each group. Appearance of IC results were shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171314#pone.0171314.s003" target="_blank">S3 Fig</a></b>.</p

List of cervical cancer-related disease groups and control groups.

<p>List of cervical cancer-related disease groups and control groups.</p