Abstract
Overexpression of human papillomavirus (HPV) 16 oncogenic protein E6 and hypermethylation of HPV16 L1 gene are of interest as biomarkers for cervical cancer progression. Immunochromatography (IC) and enzyme-linked immunosorbent assay (ELISA) are widely used to detect target biomolecules in human. In this study, we developed IC and ELISA assays to detect viral oncoprotein E6 and L1 gene methylation of HPV16 in cervical cells, respectively. The conjugated gold coated with protein G and mouse monoclonal anti-HPV16E6+HPV18E6 was optimized and can detect as low as 312.5 nanogram of HPV16 E6 purified protein using 0.5 microgram of antibody to E6 coated on strip in phosphate Buffer/0.05% Tween 20/1% BSA, pH 7.4 as running buffer. Positive band can also be detected in protein extraction from cervical cell lines containing HPV16 at least 1x105 cells. Nonspecific band was not detected when other types of HPV positive cell line and HPV negative cell line as well as bacterial cell lysate were used in the assay. The remaining cervical cells samples obtained from routine laboratory were used to evaluate the performance of the developed assay. HPV negative samples or other high-risk HPV types other than HPV16 and 18 showed negative results. HPV16 positive samples showed 40%, 22.22%, 50%, 28.6% and 18.18% positive results by IC in squamous cell carcinoma, CIN3, CIN2, CIN1 and inflammation/ASCUS groups, respectively. HPV16 L1 gene methylation status was first detected using bisulfite treated DNA and pyrosequencing assay was used for quantification of methylation. Hypermethylation was detected in cervical cancer but was low in normal/CIN1 lesions. CpG sites 5600 and 5609 are highly methylated compared to other CpGs. Indirect and sandwich ELISA were developed to detect HPV16 L1 gene methylation status using specific oligonucleotide probe for HPV16, however, it was not successful in developing sandwich ELISA assay. Indirect ELISA showed high background when oligonucleotide probe was present with other reagents in the absent of HPV16 positive DNA. Other system without oligonucleotide probe in the present of all reagents revealed low background. Optimization of blocking reagents, oligonucleotide probe concentration, streptavidin concentration, anti-5-methylcytosine dilution and incubation time were not successfully reduced the background. The ELISA assay was not yet developed successfully, however, troubleshooting will be further performed to reduce the background and can be further tested for specificity and sensitivity and will be applied in clinical samples
