Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for mitozantrone

A mouse monoclonal antibody (NO-1) with specificity for the anti-cancer drug mitozantrone (MZ) (Novantrone) was produced by immunization of a BALB/c mouse with mitozantrone-keyhole limpet haemocyanin (MZ-KLH) conjugate. When used in an indirect competitive enzyme-linked immunosorbent assay (ELISA), NO-1 permitted the accurate and reproducible detection of between 0.25-50 ng/ml of MZ in pooled human serum, the standard curve obtained within this range being virtually linear. The assay demonstrated good reproducibility with intra-assay coefficients of variation (CV) of between 1.41% and 7.02% and an inter-assay CV of 3.45%. Regression analysis of levels of MZ detected by ELISA vs. the actual amounts added to pooled human serum gave a very good correlation coefficient of r = 0.995. NO-1 showed no cross-reactivity with either bisantrene or daunorubicin. A simple pharmacokinetic study was undertaken in rabbits given MZ intravenously at a dose of 0.5 mg/kg of body weight. Levels of MZ in rabbit serum measured with the assay ranged between 82 and 170 ng/ml for rabbits 1 and 2, respectively at 15 min falling to 1.25 ng/ml by 48 h for rabbit 1 and falling to undetectable levels by 120 h for rabbit 2.