title: Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18.
abstract: A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and M. T. Esser, Clin. Diagn. Lab. Immunol. 10:108--15, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.
authors.affiliation: Vaccine and Biologics Research, Merck Research Laboratories, 466 Devon Park Dr., Wayne, PA 19087-8630, USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Dias
authors.forename: Jeff
authors.initials: J
authors.lastname: Van Doren
authors.forename: Sonela
authors.initials: S
authors.lastname: Schlottmann
authors.forename: Sheri
authors.initials: S
authors.lastname: Kelly
authors.forename: Derek
authors.initials: D
authors.lastname: Puchalski
authors.forename: Wanda
authors.initials: W
authors.lastname: Ruiz
authors.forename: Patricia
authors.initials: P
authors.lastname: Boerckel
authors.forename: Joseph
authors.initials: J
authors.lastname: Kessler
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Antonello
authors.forename: Tina
authors.initials: T
authors.lastname: Green
authors.forename: Martha
authors.initials: M
authors.lastname: Brown
authors.forename: Judith
authors.initials: J
authors.lastname: Smith
authors.forename: Narendra
authors.initials: N
authors.lastname: Chirmule
authors.forename: Eliav
authors.initials: E
authors.lastname: Barr
authors.forename: Kathrin U
authors.initials: KU
authors.lastname: Jansen
authors.forename: Mark T
authors.initials: MT
authors.lastname: Esser
mesh_terms: D000293:Adolescent; D000328:Adult; D000911:Antibodies, Monoclonal / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D002648:Child; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008297:Male; D008863:Microspheres; D027383:Papillomaviridae / Q000145:classification / Q000276:immunology*; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014412:Tumor Virus Infections / Q000175:diagnosis / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes