At each follow-up visit, one cervical sample was taken for cytological examination, followed by the collection of another sample which was stored for subsequent virological examination. the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay. Cervical human papillomavirus (HPV) infection is a common sexually transmitted disease, and infection with high-risk types is now considered a necessary cause of cervical cancer. The kinetics of the humoral immune response following an incident HPV infection in na?ve women are not well defined. There are two major difficulties. First, because cervical infection with HPV is an asymptomatic event, its onset can never be precisely defined. However, it can be approximated by making frequent observations at short intervals in women who are unexposed at recruitment. These women should be recruited soon after they have first had sexual intercourse, because the longer the interval between first intercourse and study entry, the more likely it is that a woman will have acquired and then cleared at least one HPV infection during this time (3, 9). Only two studies have fulfilled these criteria (2, 5). Both recruited university students; one included 42 women with an incident HPV type 16 (HPV-16) infection and 30 with an incident HPV-18 infection; the other included 28 women with an incident HPV-16 infection. The second difficulty relates to the assay used to measure the serological response. Both of the studies cited used virus-like-particle (VLP)-based enzyme-linked immunosorbent assays (ELISAs). The integrity of the ELISA depends on the maintenance of intact VLPs; disrupted or incorrectly folded VLPs may lead to the detection of nonneutralizing and cross-genotype-reactive antibodies, thus complicating the interpretation of the results. Competitive radioimmunoassays (cRIAs), which specifically measure HPV type-specific neutralizing antibody in serum by using mouse monoclonal YYA-021 antibodies as competitors, have also been developed. The cRIA generally has lower backgrounds than the ELISA, is more sensitive, and is less likely to be influenced by impurities. However, YYA-021 like any serological assay based on competition, it fails YYA-021 to detect serum antibodies that fail to bind to or compete with the epitopes that bind to the competing antibody. We have evaluated a neutralizing antibody assay, first described by Pastrana et al. (7), which uses HPV-16 and HPV-18 pseudovirions (PsVs) carrying a secretory alkaline phosphatase (SEAP) reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. Having first defined the reproducibility of the assay, we describe the kinetics of the neutralizing antibody response in a cohort of 42 young women YYA-021 who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. MATERIALS AND METHODS Study population. The samples used to validate this assay were collected during a prospective cohort study investigating the natural history of cervical neoplasia. The study design and the characteristics of the study population have been described elsewhere (9). In brief, 2,011 women aged 15 to 19 years were recruited from a single family planning clinic in Birmingham, United Kingdom, between 1988 and 1992 and were asked to return at intervals of 6 months; follow-up ended YYA-021 on 31 August 1997. At each follow-up visit, one cervical sample was taken for cytological examination, followed by the collection of another sample which was stored for subsequent virological examination. The women were also asked to provide a serum sample. All women with an abnormal smear, irrespective of its severity, were immediately referred Rabbit polyclonal to ZNF471.ZNF471 may be involved in transcriptional regulation to a colposcopy clinic for histological examination. Colposcopic and cytological surveillance was maintained in these women, and treatment was postponed until there was histological evidence of high-grade cervical intraepithelial neoplasia of grade 2 or 3 3, at which point the women left the study. After all clinical follow-up had ended, cervical cytology samples were tested for the presence of HPV DNA by PCR with a general primer pair (primers GP5+ and GP6+), and further PCR tests were done with type-specific primers with samples that were HPV positive after ethidium bromide staining (9). The study was approved by the appropriate research ethics committee, and informed verbal consent was obtained from all women. The measurement of intra- and interassay variations used 42 serum samples taken from 21 women in this cohort: the corresponding cytological samples had been found by PCR with primers GP5+ and GP6+ to test positive for HPV-16 DNA or HPV-18 DNA, or both, either alone or in the presence of other HPV types,.