Preparation of a specific monoclonal antibody against 2′-deoxycytidine

To evaluate the plasma 2′-deoxycytidine (2′dCyd) level as a prognostic marker for breast cancer patients, a specific monoclonal antibody was required to develop a immunoassay system for the accurate determination of plasma 2′dCyd concentration. In this study, a highly specific monoclonal antibody against 2′dCyd has been prepared. 3′-Hemisuccinyl-2′dCyd-Keyhole limpet hemocyanin protein conjugate (3′sdCyd-KLH) was used as an immunogen. WKY/NCrj rats were immunized with an emulsion of 3′sdCyd-KLH and Freundʹs complete adjuvant. A fortnight after single immunization, the medial iliac lymph-node cells were taken and fused with SP2/0 mouse myeloma cells. From the positive high titer clones, 4 clones that secreted specific anti-2′dCyd antibodies were selected. The four clones showed slight cross reactivity with 5-methyl-2′-deoxycytidine (5MedCyd) and 3′-deoxycytidine (3′dCyd). The most selective monoclonal antibody which belonged to IgG2b, κ type was termed RH-4. The specificity of RH-4 antibody in hybridoma culture supernatant was determined. Among 39 different nucleosides, nucleotides and bases tested by enzyme immunoassay (EIA), only 3′dCyd and 5MedCyd showed very slight cross reactivity. RH-4 secreting clone was grown in BALB/c slc-nude mice in vivo. After the purification of the antibody by protein A column chromatography and choice of a proper concentration, the slight cross reactivity was completely overcome. This monoclonal antibody will be useful for the determination of plasma 2′dCyd concentrations. We are now developing a sensitive and conventional method for the application of RH-4 antibody.