Development of polymerase chain reaction enzyme linked immunosorbent assay for detection of human papillomavirus 16 L1 gene methylation

Abstract

It has been well known that cervical cancer is caused by persistent infections with the high risk human papillomavirus (HR-HPV) especially HPV16 and 18. However, a minority of HR-HPV infected women developed cancer, therefore, triage test is necessary to particularly select some HR-HPV infected women who are at higher risk to progress to cervical cancer quickly for colposcopy examination. Since HPV16 L1 gene hypermethylation has been reported to be correlated well with high grade cervical lesions and cancer, the present study aims to develop PCR-ELISA for detection of HPV16 L1 gene methylation. Cervical cells were collected from women who referred for colposcopy in Chulalongkorn memorial hospital. Cobas4800 and REBA-ID assays were used to discriminate HPV 16 positive samples from other types. Of 207 samples, seventy samples resulted in HPV16 positive with histology confirmed as CIN1-3 and other abnormality. All seventy samples were used to perform bisulfite conversion and HPV16 L1 PCR to detect methylation at CpGs5600, 5606, 5609 and 5615. Twenty-six PCR positive samples (130 bps) were used in pyrosequencing assay to evaluate methylation profile which were then used as reference data for PCR-ELISA development. Methylation level >20% was detected at CpGs 5600 and 5609 of CIN2/3, while <10% methylation was mostly found in CIN1 samples. For PCR-ELISA assay optimization, different concentration of reagents was performed. Although final protocol was successfully optimized, but non-specific binding was observed when testing with 0% methylated DNA. Thus, the protocol at streptavidin and amplicons-probe hybrids binding step was changed from 37 °C to 60 °C as the same as hybridization temperature to prevent non-specific binding. We found that at adjusted protocol, the absorbance was lower in DNA concentration 10-100 ng, but still high in 1,000 ng. The final optimized protocol for further experiments was as followed: the hybridization temperature was 60◦C, 2 microgram/well of streptavidin, 294 ng/well of DIG-labelled probe and 1:3200 of antibody dilution, the binding step temperature was 60 °C. The detection limitation of CaSki DNA (approximately 100% methylation) was as low as 10 ng, however, to reduce the non-specific binding, the amplicons concentration must be lower than 1000 ng to efficiently differentiate between unmethylated and methylated CpG. It was noted that the concentrations used in standard curve controls and samples should be the same to obtain reliable absorbance for calculation of methylation percentage. The sensitivity of PCR-ELISA was as low as 20% methylation that absorbance value was higher than value obtained from 0% methylated amplicons and was consistent with pyrosequencing result (21%). Samples with 20% methylation detected by pyrosequencing assay showed methylation value 9.5% when detected by developed PCR-ELISA assay, thus, it can be indicated that clinical sample which represents methylation percentage equal or more than 10% by PCR-ELISA assay, might consider as hypermethylation sample (>20%) with CIN2+. All clinical samples’ DNA concentration was approximately 600 ng. Thus, standard DNA controls concentration 600 ng/microliter were used to set standard curves. Twenty-six samples which were previously analysed for methylation profile by pyrosequencing assay, were used to perform PCR-ELISA. Number of CIN1 and CIN2/3 samples that showed calculated PCR-ELISA methylation percentage similar to pyrosequencing assay was 71.43% (10/14) and 80% (8/10), respectively. When, percentage of methylation at 10% was used as cut-off in the present developed PCR-ELISA method, the sensitivity and the specificity would account for 50% and 71.43%, respectively. However, clinical samples used in present study is limited. Thus, the larger sample size is needed to achieve more accurate specificity and sensitivity.