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

• Published in: Analytica chimica acta 2014-12, Vol.852, p.212-217
• Main Authors: Chowdhury, Basudev, Cho, Il-Hoon, Hahn, Noah, Irudayaraj, Joseph
• Format: Article
• Language: English
• Subjects: 5-Carboxylcytosine (5caC); 5-Formylcytosine (5fC); 5-Hydroxymethylcytosine (5hmC); 5-Methylcytosine (5mC); 5-Methylcytosine - analysis; 5-Methylcytosine - blood; Blood; Cancer; Control equipment; Cytosine - analogs & derivatives; Cytosine - analysis; Cytosine - blood; DNA - blood; DNA - chemistry; DNA - genetics; Enzyme-based Immunoassay (EIA); Epigenesis, Genetic; Epigenetic modifications; Humans; Immunoassay; Immunoenzyme Techniques - methods; Limit of Detection; Lungs; Mathematical analysis; Neoplasms - blood; Neoplasms - genetics; Patients; Transformations
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2014.09.020

[Display omitted] •Development of biotin–avidin mediated enzyme-based immunoassay (EIA).•Quantitate epigenetic DNA methylated cytosine derivatives (5mC, 5hmC and 5caC) in blood samples.•Ultra-sensitive assay with enhanced limit of detection for the epigenetic marks.•First report of reduction in global 5hmC content in the blood of metastatic lung cancer.•First quantitative report of levels of 5caC in blood. 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-carboxylcytosine (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. 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. We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025±0.081, 0.023±0.006 and 0.001±0.0002, respectively. We observed a significant (p