5 ml. Ninety-six well plates were coated with HPV16, HPV18, HPV31

or HPV45 L1 VLPs (0.5 to 1.5 μg/ml) overnight at 4 °C, and blocked with 1% bovine serum albumin, 0.1% Tween-20 in phosphate-buffered saline. For the determination of the chaotropic agent concentration, coated wells were incubated with 0–8 M NaSCN for 15 min at room temperature. After a washing step, wells were incubated with biotinylated V5 (1.56 ng/ml; anti-HPV16) or J4 (6.25 ng/ml; anti-HPV18) monoclonal antibodies for 90 min at 37 °C. For the avidity ELISA, coated wells were incubated with serum samples (12 serial 2-fold dilutions) for 1 h 30 min at 37 °C. After a washing step, wells were incubated with 0 or 1 M NaSCN for 15 min at room temperature. After another washing step, wells were then incubated with biotin-conjugated anti-human IgG (Jackson; Studies 1 and 2) or Ig (Amersham; Selleck GSK2118436 Study 3). Biotinylated antibody detection used the colorimetric readout based on streptavidin-horseradish peroxidase (Amdex, GE Healthcare) and O-phenylenediamine substrate (Sigma). Optical densities were read at 492/620 nm

and antibody concentrations were calculated relative to a standard antiserum reference using SoftMaxPro software (4-parameter equation) and expressed in EU/ml. An avidity index (AI2) was calculated as a ratio of the antigen-specific antibody concentration determined after 1 M NaSCN treatment divided by the antigen-specific antibody concentration without NaSCN treatment. All statistical analyses were not part of the objectives of the Selleckchem MLN0128 clinical trials from which the samples were taken

and therefore were considered as exploratory. Parametric analyses were performed using SAS software on log10 transformed data. The Shapiro–Wilk test, Skewness and Kurtosis calculations were used to confirm normality. Differences were identified by ANOVA followed by Tukey’s test. All comparisons were two-tailed. Pearson’s r statistic was used to identify correlations between (log10 transformed) AIs and antibody concentrations. Significance was ascribed to p-values <0.05 (and in the case of antibody concentrations, to ≥2-fold differences). STK38 AIs are described to two-significant figures in the text. The HPV16 L1 and HPV18 L1 conformational epitopes that are important epitopes for neutralising antibodies [7] and [26], were evaluated in an ELISA using monoclonal antibodies V5 and J4, respectively. Both epitopes were not significantly denatured by 15 min pre-incubation with <4 M NaSCN (Fig. 1). However, 10% of HPV16 L1 conformational epitopes were denatured by 2 M NaSCN. Therefore, a 15 min incubation with 1 M NaSCN in the ELISA was selected to assess the antibody avidities with serum samples from HPV-16/18 vaccine recipients. In Study 1 and 2, the AIs of HPV16 L1- and HPV18 L1-specific antibodies were assessed in samples taken from vaccinated girls and women one month post-Dose 2 (Month 2) and one month post-Dose 3 (Month 7).