Importance of antibody concentration in the assessment of cellular hypoxia by flow cytometry: EF5 and pimonidazole.

The binding kinetics of the hypoxia marker EF5 can be quantified by uptake of (14)C-labeled drug or calibrated flow cytometry using antibodies specific for drug adducts. Maximum EF5 binding is cell-line dependent and varies directly with drug exposure (area under the curve; concentration integrated over time) but inversely with pO(2) from 0 to >100 mmHg. For pimonidazole, binding is reported to be independent of the cell line and drug AUC, being zero above 10 mmHg, with an easily discriminated increase at lower pO(2). The basis for these kinetic differences is unknown, but the main experimental variable distinguishing the two marker techniques is antibody concentration ([Ab] - pimonidazole << EF5). In this study, EF5 and pimonidazole binding kinetics were compared as a function of pO(2) and antibody concentration in cells of two rat (9L and R3230) and two human (HT1080 and SiHa) cancer cell lines. For both markers, binding varied directly with AUC at all pO(2). The dynamic range of observed binding (maximum change from 0 to 76 mmHg oxygen) decreased with antibody concentration. The pO(2) dependence of binding for pimonidazole, but not EF5, varied dramatically with antibody concentration. Thus the data presented herein do not support the reported binding kinetics of pimonidazole. In particular, it is shown that the common use of antibody concentrations much lower than antigen concentrations can lead to unreliable estimations of adduct level and hence pO(2).