Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an enzyme-based immunoassay

摘要

Background: Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carbox-ylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis. Methods: To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated enzyme-based immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection.