Structural insights into antibody-antibody interactions in sandwich ELISA: implications for assay development and performance.

The enzyme-linked immunosorbent assay (ELISA) is a widely utilized immunoassay technique in biopharmaceutical drug development due to its high sensitivity and specificity. Optimizing assay performance necessitates addressing challenges that arise during method development, particularly the selection of compatible monoclonal antibody (mAb) pairs for use as capture and detection antibodies. A common approach to measure human mAb therapeutics in nonclinical studies is to use generic human-specific mAb reagents that specifically recognize human immunoglobulin constant regions. In this study, we investigated the antibody-antibody interactions for a panel of reagent mAbs that bind to distinct epitopes in the constant regions of the human IgG heavy and light chains that may contribute to diminished signal and elevated background in the sandwich ELISA format. Using both in-solution and on-plate characterization methods including hydrogen-deuterium exchange mass spectrometry (HDX-MS), native size exclusion chromatography coupled with mass spectrometry (nSEC-MS), and biolayer interferometry (BLI), we identified signal attenuation as a result of overlapping binding sites, leading to competitive or steric interference that hinders detection antibody binding. Additionally, elevated background signals were attributed to cross-reactivity between capture and detection antibodies. Furthermore, our results also reveal that antibody interactions at distinct epitopes induce different extents of steric hindrance, which significantly impacts the degree of oligomerization within the resulting product complexes. These insights enhance our understanding of the molecular mechanisms affecting assay performance and offer strategies for improving immunoassay design.