DETECTION OF HPV 16 E6 AND HPV 16 L1 PROTEINS BY LATERAL FLOW ASSAY

Abstract

Cervical cancer, the fourth leading cause of cancer death in women worldwide, is associated with the persistence of Human Papillomarus (HPV) infection especially type 16 which covered more than 50% of the cases. In this study, HPV DNA was detected in 185 (71.71%) of 258 cervical swab samples with CIN I (66.18%), CIN II (69.57%), CIN III (81.25%) and SCC, (79.10%). The prevalence of HPV 16 among HPV DNA positive samples was 28.11% (52/185) and the highest prevalence was found in SCC 49.06% (26/53). Nowadays, the screening methods for cervical cancer are based on cytology and HPV detection which are time consuming, require pathologist and special equipments. During cancer development, high expression of E6 oncoprotein is demonstrated while the presence of L1 protein implies the productive stage. Thus detection of HPV 16 E6 might be helpful for early detection of cancer development and that of L1 protein helps in determining the stage of viral infection. Since the lateral flow assay is simple, rapid, cost-effective and no instrument requirement, in this study, a lateral flow assay based on sandwich immunochromatography combining gold nanoparticle-antibody conjugates was developed to detect HPV 16 E6 and L1 proteins. Recombinant HPV 16 E6 and L1 proteins were expressed and used as positive controls in the lateral flow assay. The lateral flow assay for detecting HPV 16 L1 was unsuccessfully developed because of the presence of urea. The developed HPV 16 E6 protein test has been only established. The antibody conjugated with AuNPs (1-10 µg/ml gold), sizes of AuNPs (10 and 40 nm), type of antibodies (polyclonal and monoclonal), and blocking solution (3% PEG and 10% BSA) were optimized. Finally, the mouse monoclonal anti-HPV 16 E6 conjugated AuNPs at the concentration of 10 µg/ml gold, rabbit polyclonal anti- HPV 16 E6 at the captured test line and goat polyclonal anti-mouse at the control line demonstrated the optimal condition providing the signal within 15 min observed by naked eyes. The 40-nm AuNPs gave better sensitivity of detection (5 µg) than the 10-nm AuNPs (20 µg). No cross reactions were found with HPV18, 39, and 45. Although, this developed assay showed very low sensitivity and was unable to test with clinical specimens, the challenge to improve the sensitivity should be further attempted.