Abstract 7060: Semi-quantitative lateral flow immunoassay for HPV serology for detection of HPV+ oropharyngeal cancer

Abstract Background: HPV (human papilloma virus) is a leading cause of 60-70% of oropharyngeal cancer in the US. Antibodies to HPV early antigens are detectable in serum samples up to two decades prior to diagnosis, so that HPV serology are potential biomarkers for screening. We have developed a rapid colorimetric lateral flow assay (LFA) for the semi-quantitative detection of antibodies for HPV16 CE2 and HPV16 E7 antigens. Methods: Recombinant his-tagged proteins (HPV16 CE2 184-365aa and HPV16 E7) were printed separately on nitrocellulose membranes, with human IgG as a control line. Plasma samples from newly diagnosed HPV+ or p16+ oropharyngeal cases (n=38) and controls (n=46) were selected for assay development and image analysis based on known serostatus. The plasma was applied to the membranes, washed and blocked with casein, and antibodies were detected using a gold-conjugated protein G secondary antibody. A Python program was developed using the OpenCV library for image processing and the scipy.signal library was used to detect bands on the strip as peaks. The area under the curve for these peaks was calculated, with a threshold >700 unit squares. The seropositivity of the LFA was compared with laboratory-based RAPID ELISA HPV serology. Results: By RAPID ELISA, the sensitivity of CE2 serology was 84% and E7 was 78.9% at >97% specificity for the plasma samples specifically selected for the assay development (not a random or matched cohort). On the LFA, the sensitivity of CE2 serology was 52.6% and E7 was 76.3% at >97% specificity. By LFA, 89.5% of cases were positive for either CE2 or E7 serology at 95.6% specificity, with strong concordance with RAPID ELISA (κ=0.853). Conclusion: The LFA test demonstrated high concordance for detecting antibodies in plasma samples compared to RAPID ELISA, providing a promising point-of-care tool for large-scale screening of oropharyngeal cancer. Citation Format: Akshansh Kaushik, Tej Patel, Joshua Eger, Ching Wen Hou, Jennifer Blain Christen, Karen S. Anderson. Semi-quantitative lateral flow immunoassay for HPV serology for detection of HPV+ oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7060.