The majority of baseline TIgG seropositive subjects displayed antibody levels near the assay cut-off (data not shown), whereas post-vaccine levels were substantially higher in virtually all subjects. The low baseline seropositivity rates with both the cLIA and PsV NAb assays suggest that the high proportion of TIgG antibodies detected at baseline reflects low specificity of the TIgG assay, or cross-reactivity with other HPV types, such as those associated with cutaneous warts which are commonly acquired in childhood [24]. Safaeian et al. [25] observed a high HPV 16 baseline seropositive rate among 18–25-year-old women tested with the Glaxo-Smith-Kline Linsitinib HPV

16 EIA compared to the cLIA and a PsV NAb assay, and noted that agreement between the cLIA and the EIA was improved by raising the cut-off of the EIA. Brown et al. suggested that the high specificity

Selleckchem SB203580 of the cLIA may make it a more suitable assay for classifying baseline seropositivity, whereas the TIgG assay detects a broader array of HPV antibodies with high sensitivity and may be more suitable for serological follow-up of vaccinated subjects over time [15]. A modest upward adjustment of the TIgG assay cut-off would considerably reduce the number of individuals we identified as seropositive at baseline, but such an adjustment would require verification that the sensitivity of the assay for assessing post-vaccine responses would not be compromised. We demonstrated that the PsV NAb assay sensitivity can be increased by determining partial neutralization endpoints. Both NT90 and NTpartial endpoints consistently yield 2- to 8-fold higher GMTs than NT100. While only 85–86% of subjects remained seropositive for HPV 18 at 36 months by both cLIA and PsV NAb (NT100 endpoint) assays, all subjects had detectable HPV 18 neutralizing antibodies at the NTpartial endpoint. Thus, we conclude that the PsV NAb assay is more sensitive than the cLIA for detection

of anti-HPV 18. The PsV NAb assay is labour-intensive and not suitable for large-scale analyses, but it can serve nearly as a useful supplementary assay. While the determination of the PsV NAb endpoints may have a subjective component, we found that the assay is reproducible over multiple test batches and between operators (data not shown). Month 7 sera were initially tested together with baseline sera, and were later re-tested together with the 18-, 24- and 36-month sera. In nearly all cases, month 7 GMTs varied by no more than one dilution between test runs. This study has some limitations. All PsV NAb assays for this report were performed with single lots of HPV 16 and 18 PsV. PsV NAb titres could be affected by variable inter-batch packaging efficiency of the RFP reporter plasmid but GMTs can be consistently derived by calibration of PsV batches using standard sera [26] and [27].