Development of a Double-Antibody Sandwich ELISA for the Detection of HPV16 E6 Protein

Background: The HPV16 E6 oncoprotein facilitates the ubiquitin-mediated degradation of the tumor suppressor p53, constituting a pivotal mechanism underlying viral immune evasion, cellular immortalization, and ultimately, malignant transformation. This study aimed to develop a reliable detection tool for the HPV16 E6 protein. Methods: Recombinant GST-E6 and E6-His proteins were expressed and purified using a prokaryotic expression system. Female BALB/c mice were immunized with GST-E6, and two hybridoma cell lines (G11A11 and A4) stably secreting anti-HPV16 E6 monoclonal antibodies were generated via hybridoma technology. Antibody pairing experiments identified A4 and G11A11 as suitable for sandwich ELISA. The optimal detection system was established using A4 antibody at 2 μg/mL for coating, HPV16 E6-His at 5 μg/mL as the detection antigen, and G11A11-HRP at a 1:200 dilution as the detection antibody. To validate the reliability, Hacat-HPV16E6 cell lysates were tested in parallel with a commercial ELISA kit. Results: After purification, the titers of both antibodies reached 1:204,800. The lower limit of quantification (LOQ) was 4.79 ng/mL and the limit of detection (LOD) was 3.39 ng/mL. The comparison with the commercial kit showed good consistency, with percentage differences ranging from 20% to 40%, confirming that the established ELISA is reliable for quantitative detection. Conclusions: This study successfully yielded high-titer and highly specific anti-HPV16 E6 monoclonal antibodies and developed a specific double-antibody sandwich ELISA, thereby furnishing a technical foundation for both fundamental research and laboratory-based applications related to HPV16-associated tumors.