Enzyme-linked immunosorbent assay for 2-deoxycytidine

An enzyme-linked immunosorbent assay (ELISA) method that can be used to qua ntify 2'-deoxycytidine (dCyd) in plasma is described. In this method, 3'-su ccinyl-dCyd bovine serum albumin conjugate (3'sdCyd-BSA) adsorbed on the mi crotiter plate wells is bound to anti-dCyd monoclonal antibody in inverse p roportion to free dCyd in the sample or standard. Bound antibody is quantif ied with alkaline phosphatase-labeled anti-immunoglobulin (Alp-IgG) antibod y and p-nitrophenylphosphate (PNPP) substrate solution. A linear relationsh ip with good correlation coefficients was obtained in the linear range of 1 0-1000 mu M. There was no cross-reactivity from structurally related compou nds with the antibody. In addition, the constituents of plasma do not affec t the assay. The intra- and inter-assay reproducibilities are satisfactory at 2-3% relative standard deviation. A short period of incubation at 37 deg rees C is required for equilibration of the antigen-antibody reaction. Ther e is a good correlation between the results obtained with the proposed ELIS A and a liquid chromatographic method over the entire linear range. The ana lytical procedure is convenient, and one can analyze 150 samples per workin g day, facilitating the processing of serial samples. The present method is expected to contribute to further elucidation of the role of dCyd in vario us biological and biochemical systems. (C)2000 Elsevier Science B.V. All ri ghts reserved.