suggests that natural HPV contamination elicits a low-avidity and less qualitative serum antibody response than HPV vaccination (Scherer et al., 2016), which might also be the case for locally-produced antibodies after natural contamination. between total human IgG concentrations in FVU samples were found for the two total IgG assays (rs 0.42, p < 0.0001), while correlations for serum were non-significant. In conclusion, the HPV16-DELFIA assay is usually usable for detecting HPV16-specific antibodies in FVU and serum samples. As total human IgG remains an interesting VEGFA parameter for the normalization of HPV-specific IgG in FVU, the accuracy of both assays needs to be validated further. Keywords:First-void urine, Human papillomavirus, HPV, Vaccination, Antibody, Self-sampling == Highlights Chlorzoxazone == HPV16 DELFIA assay is usually a suitable immunoassay for the detection of HPV16 antibodies. HPV16-specific DELFIA has high sensitivity in first-void urine and serum samples. DELFIA sensitivity is similar to existing VLP-based immunoassays M4- and M9ELISA. Normalization of antibody concentration with total IgG did not have an added value. == 1. Introduction == Human papillomavirus (HPV) vaccination is usually a crucial step in primary cervical malignancy prevention, given the direct association of prolonged high-risk HPV contamination with nearly all cases of cervical malignancy (Walboomers et al., 1999). Currently, assessing the immunogenicity of HPV vaccines predominantly relies on serum samples (Struyf et al., 2015). Interestingly, recent studies have exhibited that humoral immune responses specific to HPV can be assessed in first-void urine (FVU) (Pattyn et al., 2020a,Teblick et al., 2023,Van Keer et al., 2019). Considering that cervical cancers after HPV contamination predominantly develop at the cervical transformation zone, evaluating antibody-mediated immune responses at the cervix, and thus local protective immunity is important (Longet et al., 2011,Stanley et al., 2012). Vaccine-induced circulating antibodies reach the female genital tract (FGT) through transudation or exudation at microlesion sites. This results in a higher presence of immunoglobulins (IgG) originating from serum, with fewer locally produced IgG and secretory IgA (sIgA) (Mestecky et al., 2005,Scherpenisse et al., 2013). These HPV-related immunological markers, together with discharged mucus and (debris from) exfoliated cells from the Chlorzoxazone female genital organs, accumulate round the urethral opening and the small labia, and are subsequently washed away during urination. The initial a part of a urine sample, known as first-void urine contains a higher concentration of FGT secretions compared to the subsequent part. This includes HPV DNA, HPV-type-specific antibodies, and other HPV biomarkers (Bober et al., 2021,Pathak et al., 2014,Van Keer et al., 2022,Van Keer et al., 2021,Vorsters et al., 2014,Zhou et al., 2018). The non-invasive nature of this sample type makes it highly acceptable and allows women to collect it at home, eliminating the need for a blood draw at a medical facility (De Pauw et al., 2021). As a correlate of protection for HPV infections remains undetermined, the detection of HPV-specific antibodies at the site of contamination could emerge as an important immunological monitoring tool (Falcaro et al., 2021,Gillison et al., 2008,Longet et al., 2011,Paavonen et al., 2009,Villa et al., 2006). Recent studies have exhibited the quantifiability of HPV-specific IgG in FVU samples, revealing a significant difference in HPV-type-specific antibody concentrations between FVU samples from vaccinated and unvaccinated individuals (Pattyn et al., 2020a,Teblick et al., 2023). Given that HPV-type-specific antibody concentrations are approximately three logs higher in serum compared to FVU samples, the concentrations in FVU often reach the detection limit of existing immunoassays, which are exclusively validated for serum. Furthermore, to account for the lower HPV-specific antibody concentrations present in FVU, lower dilutions must be used, potentially leading to more non-specific background compared to serum. To improve HPV-specific antibody detection in FVU a highly sensitive immunoassay is required. Previous studies were already able to show HPV-specific antibody positivity Chlorzoxazone for HPV16 in more than 80 % of FVU samples from vaccinated women (Pattyn et al., 2020a,Teblick et al., 2023)..