Binding and inhibition assays to assess the specificity of rituximab reactivity with ASMLPD-derived peptides. (A-B) Fifty microliters of different concentrations of rituximab (A) and infliximab (B) was added to wells of a 96-well microtiter plate previously coated with pASMLPD (▪) and rev-pASMLPD (□) and incubated for 4 hours at 25°C. Peptide-antibody binding was detected with HRP-xeno-antibodies to human IgG (Fc portion). Binding of mAb to Rp15-C (○) and Qp1a (×) was included as specificity control. (C-D) Different concentrations of pASMLPD (▪), rev-pASMLPD (□), and Rp15-C (○) were mixed with an appropriate dilution of purified rituximab (5 μg/mL). After a 1-hour incubation at 4°C, the mixture was added to wells of a 96-well microtiter plate previously sensitized with peptide pASMLPD (C) and rev-pASMLPD (D). Rituximab-peptide interaction was determined with HRP-conjugated xeno-antibodies to human IgG (Fc portion). Binding of rituximab to peptide in the presence of peptide Qp1a (×) was included as negative control. Results are expressed as percentage of binding compared with binding in the absence of inhibitor.