Time-resolved fluoroimmunoassay of 5-methyl-2'-deoxycytidine employing europium-labeled antigen as tracer.

We describe a solid-phase fluoroimmunoassay, based on competition between europium-labeled 5 MeCyd (5-methylcytidine) and sample 5MedCyd (5-methyl-2'-deoxycytidine) for polyclonal anti-5MedCyd antibodies (rabbit). Europium labeling of antigen was performed using a novel polylysine-5MeCyd conjugate. Standard and sample preparations containing 5MedCyd inhibited the binding of the europium-labeled 5MeCyd to the antibody molecules. A second antibody, directed against rabbit IgG, was coated on the solid phase, and bound the IgG-5MeCyd-polylysine-europium complex, giving rapid and complete separation of antibody-bound and free antigen. The measuring range was from 3.7 to 2500 pmol of 5MedCyd per assay. A good correlation between the results obtained with TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in various DNA samples, enzymatically hydrolyzed to their constituent deoxyribonucleosides. This new TR-FIA possesses the same advantages (high sensitivity, wide assay range, rapidity, simplicity, and low cost) as the previous assay developed in our laboratories. The superiority of the new system is based on (i) its low inter- and intra-assay variation, (ii) low antiserum consumption, and (iii) a protocol, which permits the use of second-antibody-coated microtitration strips common to other assays.