Use of the Normalized Absorbance Ratio as an Internal Standardization Approach To Minimize Measurement Error in Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Papillomavirus Infection

Abstract

The serological detection of antibodies against human papillomavirus (HPV) antigens is a useful tool to determine exposure to genital HPV infection and in predicting the risk of infection persistence and associated lesions. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for seroepidemiological studies of HPV infection but are not standardized. Intra-and interassay performance variation is difficult to control, especially in cohort studies that require the testing of specimens over extended periods. We propose the use of normalized absorbance ratios (NARs) as a standardization procedure to control for such variations and minimize measurement error. We compared NAR and ELISA optical density (OD) values for the strength of the correlation between serological results for paired visits 4 months apart and HPV-16 DNA positivity in cervical specimens from a cohort investigation of 2,048 women tested with an ELISA using HPV-16 virus-like particles. NARs were calculated by dividing the mean blank-subtracted (net) ODs by the equivalent values of a control serum pool included in the same plate in triplicate, using different dilutions. Stronger correlations were observed with NAR values than with net ODs at every dilution, with an overall reduction in nonexplained regression variability of 39%. Using logistic regression, the ranges of odds ratios of HPV-16 DNA positivity contrasting upper and lower quintiles at different dilutions and their averages were 4.73 to 5.47 for NARs and 2.78 to 3.28 for net ODs, with corresponding significant improvements in seroreactivity-risk trends across quintiles when NARs were used. The NAR standardization is a simple procedure to reduce measurement error in seroepidemiological studies of HPV infection.Serological testing for circulating anti-human papillomavirus (HPV) antibodies has potential clinical utility in measuring cumulative exposure to HPV infection and as a marker of HPV-associated disease, particularly for the anogenital tract (10). Long-term persistent HPV infection tends to yield polyclonal immunoglobulin G (IgG) serological reactivity, which can be measured by using enzyme-linked immunosorbent assay (ELISA) techniques based on type-specific L1-only (7) or L1-plus-L2 virus-like particles (VLPs) as antigens (8, 11). Serological testing for HPV antibodies cannot replace direct HPV DNA measurements or cytological and histological examinations of the target tissue, but it can serve as an adjunctive tool, particularly in molecular epidemiology studies of the natural history of HPV infection and cervical precancerous lesions (3,16,17,19). Thus far, however, HPV serology has not been used in the clinical setting because of a variety of technical and biological limitations (8,10). Although the specificity of HPV VLP ELISA is reasonably adequate, at over 90%, its sensitivity is relatively low (50% to 60%) (14).Among the main technical issues that affect its clinical utility are the lack of cross-laboratory standardization and reproducibility. Aside from continued improveme...

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“…As a consequence, the normalized absorbance ratios represent multiples of reactivity in relation to the control group. 25 To analyze the lesion size, parasite burden, and immunological response between the groups, the two-way ANOVA and the area under the curve, followed by Bonferroni's post-test, were used. Differences were considered statistically significant when P0.05.…”

Estimates of Mumps Seroprevalence May Be Influenced by Antibody Specificity and Serologic Method

“…Background absorbance values measured from the untransfected Vero antigen control were subtracted from the absorbance measured from the NP and HN lysates. To normalize the antigen-specific ELISA measurements across experiments, we divided the background-corrected absorbance values of the test sera by the background-corrected absorbance values of a positive-control serum that was included with each experiment (38). Mumps NP-specific IgM was measured by ELISA using a method similar to that described for measles (39).…”

Human Papillomavirus Infection and Reinfection in Adult Women: the Role of Sexual Activity and Natural Immunity

“…An aliquot from this pool was thawed and processed in the same manner as all study serum samples included in each ELISA run and the same serum pool was used throughout the study. Absorbances were corrected for the fluctuation in seroreactivity of this serum pool as previously described (19). …”