Detection of antibodies to HPV vaccine types using a multiplexed immunoassay

Measurement of HPV antibodies in unvaccinated individuals has been used as measure of lifetime exposure to HPV. With the implementation of HPV vaccines, reliable assays are needed to evaluate the impact of altered dosing schemes or of new vaccine formulations. An ideal assay platform would allow multiplex type‐specific detection with minimal sample requirement. We used the Meso Scale Discovery (MSD) electrochemiluminescence ECL based detection platform to develop a multiplex direct‐ virus‐like particles (VLP) assay. HPV 6, 11, 16, and 18 VLPs were produced in mammalian cell‐culture and purified using Optiprep™ gradient followed by agarose gel filtration. MSD prepared the plates in the 7‐spot/well format, using the purified VLPs (4 spots) and PBS pH 7.4 as blank (one spot). We used four different coating conditions, varying VLP concentrations with and without bovine serum albumin (BSA). Results were evaluated using a set of 17 sera, WHO International Standard for HPV 16, and an optimized ELISA protocol for MSD plates on the SI6000 imager. Three point titrations and the parallel line method was used to calculate antibody titers. Stability of the plates was evaluated by comparing results after varying times of storage. The use of BSA in spot‐coating allowed for increased stability of the VLPs on the plate as indicated by similar RLUs on plates tested 1 month apart. The intra and inter‐assay CVs between PLL values calculated for each type was less than 8% and 17% respectively. No cross‐reactivity was observed between HPV types. The MSD platform shows promise for simultaneous quantitation of the antibody responses to several HPV types in a high‐ throughput manner. Funding for this project was provided by the American Recovery and Reinvestment Act. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency