A strategy was developed for the control, standardization, and critical evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of human papillomavirus-specific immunoglobulin G in human sera. Control human sera, polyclonal animal sera, and monoclonal antibodies were used to establish optimal assay parameters, including antigen coating, serum dilutions, and criteria for daily reproducibility, monitoring, and rejection of assays. Three evaluation techniques were used in parallel to define an optimal cutoff absorbance value that yields greater than 93% sensitivity and 98.5% specificity in the assay's ability to discriminate positive and negative control sera. This strategy provides an optimal method by which to determine cutoff absorbance values for ELISA.Detection of human papillomavirus (HPV) DNA in cervical samples has been considered a hallmark for the diagnosis of infection. However, since the detection of HPV DNA is often transient (2), measurement of virus-specific immunity may be a better indicator of HPV exposure since a prior or current infection is likely to be detected. Enzyme-linked immunosorbent assays (ELISA) based on the use of viruslike particles (VLPs) have been described (3, 4). However, many studies using serologic VLP ELISAs have provided insufficient details for laboratories to implement, monitor, and control assays. For example, while some studies use a variety of methods to determine a cutoff value (COV), others rely solely on COVs determined in previous studies (8-13). Due to variations in technique related to antigen lot, environmental conditions, and operator, COVs generated by ELISA may vary from study to study. To minimize such discrepancies, control and standardization methods and multiple evaluation techniques can be used to determine an optimal ELISA COV for each study independently. This report describes a method for VLP ELISA optimization, standardization, and quality control. MATERIALS AND METHODSSerum samples. A total of 109 (43 positive, 66 negative) serum samples were used as controls. Human serum samples were kindly provided by Mike Hagensee at Louisiana State University, by Howard Strickler at the National Cancer Institute (currently at the Albert Einstein School of Medicine), and by Egleston Children's Hospital (Atlanta, Ga.). Sera obtained from Louisiana State University and the National Cancer Institute were received along with an indication of their reactivity (positive or negative) to HPV type 16 (HPV-16) L1 VLPs as determined in the laboratory of origin. For negative sera obtained from Egleston Hospital, Western blotting and our ELISA were used to determine the reactivity of children's sera to HPV-16 L1 VLP antigen.In addition, both preimmune rabbit sera and rabbit anti-HPV-11 L1-specific, anti-HPV-18 L1-specific, anti-HPV-31 L1-specific, and anti-HPV-16 L1-specific sera were kindly provided by Robert Rose, University of Rochester Medical Center (University of Rochester, Rochester N.Y.). Monoclonal antibodies (MAbs; kindly provided by Niel Christen...
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