Fig. 1: Process flow diagram for testing, selection and formulation of donations from naturally infected women to produce the 7 candidate WHO International Standards for HPV antibodies.

Twenty anonymized donations obtained from women naturally infected with Human Papillomavirus (HPV) were provided for initial testing in HPV type-specific pseudovirion-based neutralization assays (PBNA) and antibody binding (Ab-binding) assays. Thirteen candidate samples shown to be seropositive for the target HPV types were selected for further development. Candidate samples for antibodies to HPV6, HPV31, HPV33, HPV45, HPV52 and HPV58 were selected for optimization of pooling ratios to obtain lowest possible cross-reactivities for non-target HPV types. The HPV11 candidate samples were pooled without optimization. The candidate samples were then filled into ampoules and freeze-dried in separate manufacturing procedures to produce the 7 candidate International Standards. Prior to their formal assessment in the multicenter international collaborative study, the candidate International Standards underwent validation testing in PBNA and Ab-Binding assays. The single asterisk indicates that seronegative serum was used for optimizing the reactivities of candidate samples for HPV31 and HPV45 antibodies. The double asterisk indicates that pooling of candidate samples for HPV11 antibodies was not optimized based on the exception criteria of “no type-cross-reactivity” due to difficulty in sourcing monospecific material. The triple asterisk indicates that the candidate International Standards for HPV31 and HPV45 antibodies were validated after the optimization procedure. The candidate International Standards for HPV6, HPV11, HPV33, HPV52 and HPV58 were validated after freeze-drying.