Characterization of the B-B4 mAb. (A) The affinity of B-B4 mAb and the number of antigenic sites on MDA-MB-468 breast cancer cells were evaluated by an equilibrium-binding assay with 125I-labeled B-B4 mAb. The nonspecific binding of 125I-labeled B-B4 mAb was evaluated with a tenfold excess of unlabeled B-B4 mAb at three concentrations of 125I-labeled B-B4 mAb and subtracted from binding data. The mean results of a triplicate assay are represented. The Bmax corresponding to the number of antigenic sites on MDA-MB-468 cells, and the Kd of 125I-labeled B-B4 were determined by nonlinear regression using a one-binding site equation. (B) The immunoreactivity of 125I-labeled B-B4 mAb was estimated by measuring the binding of a constant concentration of 125I-labeled B-B4 mAb to increasing numbers of U266 multiple myeloma cells that strongly expressed the CD138 antigen. This is a double-inverse plot of a triplicate assay. The fraction of immunoreactive 125I-labeled B-B4 (R) was determined as the inverse of the ordinate at the origin of the abscissa according to the Lindmo method .