title: Development and evaluation of multiplexed immunoassay for detection of antibodies to HPV vaccine types.
abstract: Reliable antibody based-assays are needed to evaluate the immunogenicity of current vaccines, 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 based detection platform to develop a multiplex direct virus-like particle (VLP) ELISA to detect antibodies to HPV 6, 11, 16, and 18 with a protocol developed for detection using the SI 6000 imager (M4ELISA). MSD prepared the plates in the 7-spot/well format, using the purified VLPs (4 spots) and PBS+BSA pH7.4 (3 blank spots). Three-point titrations and the parallel line method were used to calculate antibody levels. Dynamic range, precision, and stability of pre-printed plates were determined using a panel of previously characterized sera. Cut-off values using children's sera were established using 99% RLU limits based on the 4-parameter Johnson Su best fit curve. Results of the M4ELISA were compared to competitive Luminex Immunoassay (cLIA) on n = 4454 sera from a predominantly unvaccinated cohort. Using a VLP coating concentration of 80 μg/ml with BSA provided the most robust RLU signal for all types. The dynamic range of the assay was about 1000 fold, with assay variability under 25% for each of the four vaccine types. Long-term stability of the plates extended to about 7 months from the time plates was received in the laboratory after printing. There was moderate agreement (κ = 0.38-0.54) between M4ELISA and cLIA, with antibody detection for each of the 4 types more frequent with M4ELISA. Quantitative analysis however showed a good correlation between concordant samples by both assays (ρ ≥ 0.6). The MSD platform shows promise for simultaneous quantitation of the antibody responses to four HPV vaccine types in a high-throughput manner.
authors.affiliation: Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, USA.
authors.forename: G
authors.initials: G
authors.lastname: Panicker
authors.affiliation: Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, USA.
authors.forename: I
authors.initials: I
authors.lastname: Rajbhandari
authors.affiliation: Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, USA.
authors.forename: B M
authors.initials: BM
authors.lastname: Gurbaxani
authors.affiliation: Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Querec
authors.affiliation: Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, USA.
authors.forename: E R
authors.initials: ER
authors.lastname: Unger
mesh_terms: D000914:Antibodies, Viral / Q000097:blood*; D002648:Child; D055664:Electrochemical Techniques / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D052140:Human papillomavirus 11 / Q000276:immunology; D052162:Human papillomavirus 16 / Q000276:immunology; D052161:Human papillomavirus 18 / Q000276:immunology; D052163:Human papillomavirus 6 / Q000276:immunology; D006801:Humans; D008297:Male; D053918:Papillomavirus Vaccines / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D053918:Papillomavirus Vaccines
keywords: ELISA; HPV antibodies; Meso Scale Discovery; Multiplex; Serology