Preparation of a monoclonal antibody specific for 5-methyl-2′-deoxycytidine and its application for the detection of DNA methylation levels in human peripheral blood cells

Abstract

A monoclonal antibody specific for a modified nucleoside, 5-methyl-2′-deoxycytidine (m5dCyd), was prepared using 5-methylcytidine (m5Cyd)-keyhole limpet haemocyanin (KLH) conjugate, and was characterized. Termed FMC9, the antibody reacts with m5dCyd and slightly with m5Cyd and 5-methylcytosine (m5Cyt) but not with other nucleosides tested in this investigation. FMC-9 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of m5dCyd levels. Sensitivity was in the picomole range. Methylation levels in peripheral blood cells of healthy donors were determined by inhibition ELISA. The percentage of m5dCyd in peripheral blood cells of 10 healthy donors was 5.08 ± 0.50%. These results suggest that the inhibition ELISA using FMC9 is useful to monitor m5dCyd levels in the peripheral blood cells.