Serum mAb levels correlate with B-cell depletion kinetics. (A-B) WT, hCD20 Tg, and CD20 Tg γ-chain^−/− mice were treated with mAbs (250 μg), and the serum mAb concentration measured after 1, 7, and 28 days by incubating sera with SU-DHL-4 cells and then detecting cell-bound mAbs with FITC-labeled anti–mouse Fc, and comparing the level with a standard curve. (A) Representative histograms; solid histogram, background staining; blue, type II mAb tositumomab; red and green, type I mAb Rit m2a and 1F5, respectively. (B) The concentration of mAbs in the serum; n = 3 mice per group. Bars represent mean ± SD. (C) Correlation of serum mAb levels and B-cell repopulation in the periphery. hCD20 Tg mice were treated with a single dose of mAb (250 μg) and the mAb level in the serum and B-cell numbers assessed. Bars represent mean ± SD; n = 3 mice per group. (D) Repeated low-level dosing (25, 50, or 100 μg weekly for 5 weeks) with Rit m2a potentiates B-cell depletion compared with a single large dose (250 μg) of mAb to a level equivalent to that seen with tositumomab. The level of B cells in the blood and secondary lymphoid organs was assessed on day 40 by flow cytometry. T cells (blue) and B cells (yellow) in day 40 spleen sections were also visualized by confocal microscopy as described in “Methods” using an HCX PL FLUOTAR 10×/0.3 lens and 1× optical zoom. Serum mAb levels were also assessed (supplemental Figure 5) with repeated doses shown to enhance mAb serum levels.