PREPARATION OF A SPECIFIC MONOCLONAL-ANTIBODY AGAINST 2'-DEOXYCYTIDINE

To evaluate the plasma 2'-deoxycytidine (2'dCyd) level as a, prognosti c marker for breast cancer patients, a specific monoclonal antibody wa s required to develop a immunoassay system for the accurate determinat ion of plasma 2'dCyd concentration. In this study, a highly specific m onoclonal antibody against 2'dCyd has been prepared. 3'-Hemisuccinyl-2 'dCyd-Keyhole limpet hemocyanin protein conjugate (3'sdCyd-KLH) was us ed as an immunogen. WKY/NCrj rats were immunized with an emulsion of 3 'sdCyd-KLH and Freund's complete adjuvant. A fortnight after single im munization, the medial iliac lymph-node cells were taken and fused wit h SP2/0 mouse myeloma cells. From the positive high titer clones, 4 cl ones that secreted specific anti-2'dCyd antibodies were selected. The four clones showed slight cross reactivity with 5-methyl-2'-deoxycytid ine (5MedCyd) and 3'-deoxycytidine (3'dCyd). The most selective monocl onal antibody which belonged to IgG(2b), kappa type was termed RH-4. T he specificity of' RH-4 antibody in hybridoma culture supernatant was determined. Among 39 different nucleosides, nucleotides and bases test ed by enzyme immunoassay (ELA), only 3'dCyd and 5MedCyd showed very sl ight cross reactivity. RH-4 secreting clone was grown in BALB!e sie-nu de mice in vivo. After the purification of the antibody by protein A c olumn chromatography and choice of a proper concentration, the slight cross reactivity was completely overcome. This monoclonal antibody wil l be useful for the determination of plasma 2'dCyd concentrations. We are now developing a sensitive and conventional method for the applica tion of RH-4 antibody. (C) 1998 Elsevier Science B.V.