Description of a novel multiplex avidity assay for evaluating HPV antibodies

Limited data exists regarding antibody avidity for human papillomavirus (HPV). We describe development of a multiplex electrochemiluminescent avidity ELISA for four HPV types (HPV 6, 11, 16, 18) by adding a dissociating step to our established multiplex HPV VLP ELISA. Initial experiments exploring ammonium thiocyanate, sodium thiocyanate and guanidine hydrochloride (GuHCl) as dissociating agents identified GuHCl as most promising. Dissociation conditions with GuHCl were varied (concentration, incubation time, temperature) to select conditions with minimal impact on VLP integrity as measured with monoclonal antibodies to conformational epitopes. Avidity index (AI) was calculated based on a standard curve as ratio of bound IgG in GuHCl treated versus untreated sample. To evaluate our assay we determined AI in sera with known HPV titers. We selected 32 residual anonymized sera from individuals with a wide range of titers for HPV6, 11, 16, and 18. AIs were similar across multiple dilutions of serum within the assay's dynamic range and were reproducible with two plate lots. This assay will aid in understanding HPV antibody avidity and maturation in response to natural infection and varying vaccine schedules. This is the first report of a VLP-based multiplexed avidity ELISA that evaluates assay parameters for all nine HPV vaccine types.