An immunoassay based on polyHRP signal amplification for HPV16/18 E6 RNA detection with anti-DNA-RNA hybrid antibody.

Human papillomavirus (HPV) types 16 and 18 are major causes of cervical cancer, with their E6 oncogenes serving as key biomarkers for early diagnosis. Traditional molecular detection methods, such as PCR, are commonly used but still have limitations, including false positives and complex procedures. To address these challenges, we developed an amplification-free sandwich direct enzyme-linked immunoassay (sELISA). This assay combined the S9.6 antibody's specificity for DNA-RNA hybrids with streptavidin-polymeric horseradish peroxidase (SA-polyHRP) for signal enhancement. The SA-polyHRP direct sELISA achieved a detection limit of 1.03 pM (0.012 pg/µL), demonstrating 59.6-fold higher sensitivity than the indirect sELISA using SA-coated plates with HRP-labeled secondary antibody (monomeric HRP) for tracer. The integral evaluation of immunoassays containing specificity, affinity, and robustness was accomplished by assessing selectivity for nucleic acid structure, length, GC content variations, and subtypes, respectively. Additionally, the assay was successfully applied to artificial plasma samples, showing acceptable recovery rates (76-108%) at various dilutions and further validating its potential for clinical diagnostics. Overall, this assay using the SA-polyHRP-based direct sELISA provided a rapid, highly specific, and sensitive diagnostic tool for the detection of HPV-related cancers, which will offer significant improvements in diagnostic accuracy, screening, and monitoring.