Inhibition assay to define the spatial relationship between the peptide-specific epitopes and the rituximab antigen-combining site. (A) Fifty microliters of a PBS solution containing 2-fold serial dilutions of Rp1-L (♦), Rp5-L (▴), Rp15-C (○), Rp3-C (▵), mRp3-C (□), and RpCD20-L (▪) was mixed with an equal volume of an appropriate dilution of biotinylated rituximab. After 2-hour incubation at 4 °C, the mixture was added to target Raji cells (7 × 105 cells/well) and their binding to rituximab was detected with HRP-avidin. Binding of rituximab in the presence of unlabeled rituximab (+), infliximab (•), and peptide Qp1a (×) were included as specificity controls. Results are expressed as percentage inhibition of binding compared with binding in the absence of inhibitor. (B) One microliter of PBS solution containing 10-fold serial dilution of Rp1-L (♦), Rp5-L (▴), and Rp15-C (○; starting concentration 1 mg/mL) was mixed with 9 μL PBS solution containing rituximab (500 ng/mL). After 1-hour incubation at 4°C, the mixture was added to Raji cells (1 × 104 cells/well). Following 30-minute incubation at 25°C, an appropriate dilution of rabbit complement was added to each well and the assay was continued as described in “CDC assay.” Lysis mediated by rituximab in the presence of infliximab F(ab′)2 (•) and mAb HC-10-specific peptide Qp1a (□) was included as negative control. Lysis mediated by rituximab in the presence of rituximab F(ab′)2 (+) was used as positive control.
