Binding of Chicken anti Bevacizumab idiotype to human IgG1

Figure 1, Binding of anti-Bevacizumab is only to the idiotype and not the whole IgG molecule. Anti Bevacizumab idiotype was tested for binding to Bevacizumab and human IgG1 by indirect ELISA. Bevacizumab or human IgG1 was coated onto ELISA plates at equivalent concentrations and blocked using 2% BSA in PBS. Chicken anti-Bevacizumab idiotype was diluted to 15ng/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-Bevacizumab idiotype antibody is strongly specific to the Bevacizumab binding site (idiotype), and has negligible binding to the conserved regions of the IgG1 protein.

Comparing chicken anti-Bevacizumab idiotype to monoclonal anti-Bevacizumab idiotype in Bevacizumab sandwich ELISA.

Figure 2. Comparison of a chicken anti-Bevacizumab idiotype and monoclonal anti-Bevacizumab idiotype for use in Bevacizumab capture ELISA. Each antibody (chicken anti-Bevacizumab idiotype vs. monoclonal antibody) was coated onto polystyrene ELISA plates at equivalent concentrations for comparison in a Bevacizumab capture ELISA. Bevacizumab was spiked into human serum at concentrations from 50ng/ml down to 1.56ng/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-Bevacizumab idiotype antibody performs better as a capture antibody than the competing monoclonal antibody and provides a lower limit of detection.