Evaluation of semi-homogeneous assay formats for dual-specificity antibodies.

Abstract Dual specificity antibodies such as bispecific and Dual Action Fab (DAF) antibodies are emerging therapeutic products with powerful therapeutic potential. New bioassay formats are needed in order to monitor their dual biological activities. Here we describe the optimization and development of a “bridging” binding method in semi-homogenous (SH) assay format for a bi-specific antibody. In the SH assay format, the antigen and secondary antibodies are mixed directly with the sample solution. The bound complex is then captured onto a microtiter plate coated with the other antigen and subsequently detected by colorimetric methods. We demonstrated that an SH assay achieved comparable dynamic range, quantitation, and specificity to the conventional assay format where each reagent is added sequentially followed by separate washing and incubation steps. The SH assay requires fewer wash steps and the overall assay time is shortened by half, which translates to improved efficiency without any requirement for new equipment and reagents. Using the SH assay format, we also demonstrated that a DAF antibody which can bind to two different antigens with either arm could bind both antigens simultaneously in vitro . The SH assay format has broad application as an assay platform for assessing antigen binding properties efficiently.