A highly sensitive enzyme immunoassay for evaluation of 2′-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy

Abstract

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2′-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5′-succinyl-dCyd (5′sdCyd) conjugate of bovine serum albumin (5′sdCyd–BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5′sdCyd–BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3′,5,5′-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5′sdCyd–BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of ≤10% was 20–800 nM. No cross-reactivity from the structurally related nucleobases, nucleosides, and nucleotides was observed in the proposed assay. Mean analytical recovery of added dCyd was 98–100 ± 3.2–8.2%. The precision of the assay was satisfactory; R.S.D. was 3.4–4.2 and 4.3–8.9% for intra- and inter-assay precision, respectively. The proposed EIA was compared favorably with HPLC method in its ability to accurately measure dCyd spiked into plasma samples. The analytical procedure is convenient, and one can analyze 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed EIA is expected to contribute in further evaluation of dCyd as a prognostic marker for breast cancer chemotherapy and elucidation of the role of dCyd in various biological and biochemical systems.