Binding of Chicken anti-Rituximab idiotype to Rituximab vs human lgG1

Binding of anti-Rituximab is only to the idiotype and not the whole IgG molecule. Anti-Rituximab idiotype was tested for binding to Rituximab and human IgG1 by indirect ELISA. Rituximab or human IgG1 was coated onto ELISA plates at equivalent concentrations and blocked using 2% BSA in PBS. Chicken anti-Rituximab idiotype was diluted to 0.25ug/ml and applied in triplicate to the coated wells and incubated for 1 hour at room temperature. After washing the wells, peroxidase conjugated detection antibody (Rabbit anti chicken IgY) was applied to the wells at a concentration of 5ng/ml and incubated for 1 hour at room temperature. After a final wash, the wells were developed with TMB and absorbance at 450nm was measured. These results show that the anti-Rituximab idiotype antibody is strongly specific to the Rituximab binding site (idiotype), and has negligible binding to the conserved regions of the IgG1 protein.

Comparing Chicken anti-Rituximab idiotype to monoclonal anti-Rituximab idiotype in Rituximab sandwich ELISA.

Comparison of a chicken anti-Rituximab idiotype and monoclonal anti-Rituximab idiotype (Clone 10C5) for use in Rituximab capture ELISA. Each antibody (chicken anti-Rituximab idiotype vs. monoclonal antibody 10C5) was coated onto polystyrene ELISA plates at equivalent concentrations for comparison in a Rituximab capture ELISA. Rituximab was spiked into human serum at concentrations from 200ng/ml down to 2.5ng/ml and applied to each ELISA plate in duplicate and incubated for 1 hour at room temperature. After washing, peroxidase conjugated detection antibody (anti-human IgG1) was applied to the wells at a concentration of 650pg/ml and incubated for 1 hour at room temperature. After a final wash, the wells were developed with TMB and absorbance at 450nm was measured. These results show that the chicken anti-Rituximab idiotype antibody performs better as a capture antibody than the competing monoclonal antibody (10C5) and provides a lower limit of detection.