The Influence of Covalent Immobilization Conditions on Antibody Accessibility on Nanoparticles

Bedabrata Saha, Pål Songe, Toon H. Evers, and Menno W.J. Prins

Accepted in Analyst (2017)

The accessibility of particle-coupled antibodies is important for many analytical applications, but comprehensive data on parameters controlling the accessibility are scarce. Here we report on the site-specific accessibility of monoclonal antibodies, immobilized on magnetic nanoparticles (500 nm) by the widely used covalent EDC coupling method, under variation of four key coupling parameters (surface activation and immobilization pH, crosslinker and antibody concentration ratios). By developing a quantitative radio-labelled assays, the number of immobilized antibodies, the Fab domain accessibility (in a sandwich immunoassay), and the Fc domain accessibility (in a Protein G assay) were determined. For sub-monolayer surface coverage, the observed numbers of accessible Fab and Fc domains are equal and scale linearly with antibody density. Above monolayer coverage, the fractions of accessible Fab and Fc domains decrease, in an unequal manner. The results show that the antibody accessibility is primarily determined by antibody surface density, rather than by chemical reactivity or charge state, and that crowded conditions affect Fab and Fc accessibility in an unequal manner.