Development of an ELISA-Based Pulldown Approach for Functional Analysis of Antigen-Specific Antibodies

Summary The identification of antigen-specific neutralising antibodies is essential for vaccine development and therapeutic discovery, yet current methods either lack functional readouts or are impractical for polyclonal sera from large cohorts. Here, we describe an ELISA-based pulldown methodology that isolates functional antibodies from serum samples while preserving their neutralising activity for downstream applications. We optimised elution conditions using 3M MgCl 2 in HEPES buffer, which effectively disrupts antibody-antigen interactions without dislodging immobilised antigen or impairing antibody function. The assay demonstrated 100% specificity and 77.78% sensitivity for detecting known positive samples. Compatibility with pseudotype neutralisation assays was established, with maximum tolerable MgCl 2 concentrations defined for direct use without dialysis. As proof-of-concept, we applied the method to identify domain-specific neutralising antibodies against the influenza virus haemagglutinin, distinguishing head-targeting from stem-targeting responses in human sera. This methodology provides a scalable platform for dissecting functional antibody responses with epitope-level resolution. Highlights Adapted ELISA pulldown isolates functional antigen-specific antibodies 3M MgCl 2 elution preserves antibody functionality Compatible with Influenza pseudotype neutralisation assays Allows for the identification of region-specific antibodies Graphical Abstract