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Optimization and Validation of a Multiplexed Luminex Assay To Quantify Antibodies to Neutralizing Epitopes on Human Papillomaviruses 6, 11, 16, and 18

Dennis Dias ,^1,, Jeff Van Doren,^1, Sonela Schlottmann,¹ Sheri Kelly,¹ Derek Puchalski,¹ Wanda Ruiz,¹ Patricia Boerckel,¹ Joseph Kessler,¹ Joseph M. Antonello,² Tina Green,² Martha Brown,³ Judith Smith,³ Narendra Chirmule,¹ Eliav Barr,⁴ Kathrin U. Jansen,^3, and Mark T. Esser^1*

Vaccine and Biologics Research, Merck Research Laboratories, 466 Devon Park Dr., Wayne, Pennsylvania 19087-8630,¹ Vaccine Biometrics Research,² Vaccine and Biologics Research, Merck Research Laboratories, West Point, Pennsylvania 19486,³ Vaccine and Biologics Clinical Research, Merck Research Laboratories, Blue Bell, Pennsylvania 19422⁴

Received 16 January 2005/ Returned for modification 23 February 2005/ Accepted 29 April 2005

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. 10:108-115, 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.

These authors contributed equally to this work.

Present address: Medical College of Virginia, Richmond, VA 23220.

VaxGen Inc., Brisbane, CA 94005.