Enzyme-linked immunosorbent assay for 2-deoxycytidine

Abstract

An enzyme-linked immunosorbent assay (ELISA) method that can be used to quantify 2′-deoxycytidine (dCyd) in plasma is described. In this method, 3′-succinyl-dCyd bovine serum albumin conjugate (3′sdCyd–BSA) adsorbed on the microtiter plate wells is bound to anti-dCyd monoclonal antibody in inverse proportion to free dCyd in the sample or standard. Bound antibody is quantified with alkaline phosphatase-labeled anti-immunoglobulin (Alp-IgG) antibody and p-nitrophenylphosphate (PNPP) substrate solution. A linear relationship with good correlation coefficients was obtained in the linear range of 10–1000 μ M. There was no cross-reactivity from structurally related compounds with the antibody. In addition, the constituents of plasma do not affect the assay. The intra- and inter-assay reproducibilities are satisfactory at 2–3% relative standard deviation. A short period of incubation at 37°C is required for equilibration of the antigen–antibody reaction. There is a good correlation between the results obtained with the proposed ELISA and a liquid chromatographic method over the entire linear range. The analytical procedure is convenient, and one can analyze 150 samples per working day, facilitating the processing of serial samples. The present method is expected to contribute to further elucidation of the role of dCyd in various biological and biochemical systems.