Novel competitive Enzyme-linked Immunosorbent Assay for the detection of the high risk Human Papillomavirus 18 E6 oncoprotein

CONTRERAS NE; ROLDÁN JS; CASTILLO DS

Congreso; LXVIII Reunión anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2023

Cervical cancer represents a global concern with 604,000 new cases and 342,000 deaths reported annually, with the vast majority diagnosed in low income countries. Despite high-risk Human Papillomavirus (HR HPV)-induced cervical cancer has become highly preventable through prophylactic vaccines, screening programs are critical in the control of cervical carcinogenesis in populations with limited access to vaccination and in older generations of women who have already been exposed to HR HPV infection. In this context, the E6 oncoprotein from HR HPV types arises as a promising diagnostic marker for its overexpression in transformed HPV positive cancer cells. For this reason, the aim of this study consisted of obtaining monoclonal antibodies (mAbs) against the E6 oncoprotein of one of the most prevalent HR HPV types worldwide, HPV18, in order to develop a highly specific and sensitive indirect competitive ELISA (icELISA). We selected the 7D2 hybridoma clone, which enabled the development of a sensitive icELISA to detect and quantify small amounts (226 ng/ml) of E6 disease marker. To validate our icELISA, we performed spike-and-recovery tests in C-33 A HPV-negative cervical cancer cell lysates spiked with three concentrations of HPV18 E6 recombinant oncoprotein. The average recoveries were between the ideal range from 80 to 120%. Furthermore, we carried out linearity-of-dilution assays with cells extracts from HEK293T cells that stably express HPV18 E6. Results showed an average concentration of 4.5 ng/ml of HPV18 E6 oncoprotein per 1000 cells. Finally, we tested cells extracts of HPV18-positive cervical cancer-derived HeLa cell line, which gave high signal (632.5 ng/ml). In conclusion, the present study establishes a valid, sensitive, reliable and reproducible 7D2-based icELISA that constitutes a promising bioanalytical method for the early detection and quantification of HPV18 E6 oncoprotein in cervical swab samples and cancer prevention.