Adaptation of an ELISA assay for detection of IgG_2a responses against therapeutic monoclonal antibodies in a mouse immunization model

Biotherapeutic monoclonal antibodies (mAb) play important roles in clinical medicine but their potential to elicit immune responses in patients remains a major issue. In a study designed to investigate the effect of aggregation on immunogenic responses, mice were immunized with two monoclonal antibodies (mAb1 and mAb2). Serum levels of total IgG, IgG₁, and IgG_2a were measured by ELISA. An anti-mouse IgG_2a monoclonal detection antibody cross-reacted with mAb2 but not mAb1, leading to high background when the ELISA plate was coated with mAb2. The problem was solved by use of a goat anti-mouse IgG_2a polyclonal antibody that demonstrated the required specificity. IgG_2a responses were similar for monomer- or aggregate-coated ELISA plates. The results demonstrate the importance of assessment of the specificity of individual reagents when measuring antibody responses against therapeutic antibodies by ELISA.