Validation of a pseudovirus-based neutralization assay for detection of human papillomavirus antibodies in human serum samples

Objective To validate a pseudovirus-based neutralization assay for the detection of antibodies against human papillomavirus (HPV) in human serum samples. Methods The specificity, accuracy, precision, range of linearity, limit of detection and robustness of the neutralization assay were evaluated using HPV-negative serum samples, vaccinated serum samples with quadri-valent vaccine, and international standards for detecting antibodies against HPV16 and HPV18. Results Based on the data of the HPV-naive samples, the criteria of positivity was determined as follows: the 50% inhibitory dose (ID50) of the tested sample was not less than 40 and 2-fold not less than that for bovine papillomavirus. The neutralization assay showed good accuracy with a recovery rate of 87%-122% and excellent reproducibility with intra- and inter-assay variation of 5%-27% and 10%-26% respectively. The HPV16 and HPV18 international standards were used to define the limit of quantification, which was 1.28 IU/ml for HPV16 and 0.96 IU/ml for HPV18. Acceptable ranges of variation for the key parameters of this assay were defined, which showed the good robustness of the pseudovirus-based neutralization assay. Conclusion The pseudovirus-based neutralization assay for the detection of HPV antibodies showed good specificity, accuracy, sensitivity, and robustness, suggesting that it could be used to evaluate the immunogenicity of HPV vaccines. Key words: Human papillomavirus; Neutralizing antibody; Validation