Two multiplex Luminex immunoassays are used to assess antibody responses in MSD Gardasil-9 clinical trials: (1) The primary immunoassay is the HPV 6, 11, 16, 18, 31, 33, 45, 52, 58 competitive Luminex immunoassay (HPV-9 cLIA) and (2) the secondary assay, the HPV 6, 11, 16, 18, 31, 33, 45, 52, 58 total IgG Luminex immunoassay (HPV-9 IgG assay), is used for supportive analyses. Recently, both assays were re-developed, and the optimized assays were validated and approved by the Center for Biologics Evaluation and Research, U.S. Food and Drug Administration. In addition, the assays were formally bridged to the previous assay versions to assess serostatus cutoffs (SSCO) and the impact of the changes on persistence studies.
The optimization of the assays included assessment of the following parameters: VLP coating concentration, wash buffer, Luminex microspheres, serum sample and reference standard diluent, reference standard starting dilution and titration series, and vendor and concentration of the PE-labeled antibodies. For both assays, the validation studies evaluated various performance parameters including intra-assay precision (repeatability), intermediate precision, linearity, relative accuracy/dilutability, and limits of quantitation. For the bridging study, individual patient sera from an MSD clinical trial, including day 1, month 7, and month 36 serum samples from 100 subjects, and an additional 50 day 1 samples, were used to compare measured concentration results to the historical values.
Analysis of the validation data indicates that the optimized HPV-9 cLIA and IgG assays are accurate, specific, and precise throughout the quantifiable range for each HPV type. Results of the bridging study indicate that there is a strong positive linear association between the assay versions. For both HPV-9 cLIA and IgG assays, the SSCOs were adjusted to align seropositivity rates between assay versions.
Optimization of the assay, including the elimination of antibody-depleted human serum in the assay buffer and increasing the starting dilution from 1:4 to 1:10, led to an improvement in the dilutability of the HPV-9 cLIA (within 1.25-fold per 10-fold dilution) relative to the prior version. For both HPV-9 cLIA and IgG assays, the strong positive linear association between the previous version and optimized version allow for immunogenicity assessments of long-term follow-up studies across assay versions.