Biophysical analysis of monoclonal antibodies using immunogold conjugates

Abstract

This high-throughput screening assay – which we refer to as affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) – involves first coating gold particles with capture (anti-mAb) antibodies and then using these immunogold conjugates to immobilize the target mAbs. We have used this approach to analyze large libraries of unpurified and purified mAbs to identify antibodies with a range of self-association behaviors. Importantly, our measurements of mAb self-association performed at dilute (microgram per mL) antibody concentrations are well correlated with high concentration (milligram per mL) antibody behavior. We have also developed an improved methodology for preparing stable antibody-gold conjugates that addresses the variability due to residual contaminants during gold nanoparticle synthesis. Our sedimentation-based purification methods coupled with novel acidic pH conjugation conditions yield highly reproducible and sensitive AC-SINS results. We expect our methods will be largely useful for early antibody selection, characterization and formulation. More broadly, our findings should improve preparation of stable and reproducible protein-gold conjugates for diverse applications.