A two-site enzyme immunoassay for quantitation of human papillomavirus type 16 particles

A group of human papillomaviruses (HPV), in particular HPV type 16, are the major cause of anogenital dysplasias, which are precursors of anogenital cancer. The mode of transmission, extent of infectivity and natural history of infection are incompletely understood because methods to quantify shedding of viral particles have not been available. A two-site ELISA was developed to detect and quantify HPV-16 particles. Rabbits and guinea pigs were immunized with a series of peptides from the L1 and L2 capsid proteins of HPV-16. Among rabbit antipeptide sera tested for use as capture antibodies, only sera against one peptide bound detectable amounts of virus. Guinea pig antisera against several peptides were used as reporter antibodies to detect bound virus particles. If antisera against the same peptide were used both as capture antibody and reporter antibody, only intact particles were detected. Disrupted particles were quantified using antibodies against one L1 peptide as capture antibody and antibodies against other L1 peptides as reporter antibody. The lowest detectable amount of virus was 3 ng (0.06 micrograms/ml). There was no detectable cross-reaction with HPV type 6 or 11. The assay could be used both with cervical swabs in several common sample collection buffers and with surgical material solubilized in NP40-containing extraction buffers. Among 15 surgically removed condyloma acuminata, only 1 specimen was found to contain HPV-16 particles, at a concentration of 375 ng/ml (1.1 micrograms/specimen). Among 29 cervical swab samples from patients with koilocytotic atypia, 9 samples were found to contain virus. The results indicate that this assay is useful for large-scale studies on shedding of HPV particles.