Strategies for the Immobilization of Antibodies

Abstract

The immobilization of antibodies in two-dimensional (physical adsorption and immobilization via binding proteins) and that in three-dimensional (chemical binding to polypeptide strands) coverage of solid matrices have been compared. Methods of optimizing exposure of the immunoglobulin paratope after binding to surfaces to permit unimpaired antibody-antigen complex formation have been investigated. At comparable antibody densities, a larger number of paratopes are available for complex formation when antibodies are immobilized by means of binding protein (Protein A, Protein G, secondary antibodies, and F(ab) 2 fragments of secondary antibodies) compared to physical adsorption. Using a primary coat of binding protein, reproducible immobilization of antibodies at densities up to two orders of magnitude lower than densities produced by physical adsorption and chemical immobilization is achieved. Binding of antibodies by means of polypeptide strands can be used to obtain multi-layers of IgG molecules. This method of immobilization gives the IgG molecules some flexibility in lateral positioning which leads to formation of clusters and increased attachment to the solid matrix, impairing the paratopes′ ability to form complexes. However, flexible attachment can permit separation of the IgG molecules by treatment with detergents (e.g., Tween) or with charged molecules (e.g., fluorescein) or by introducing binding proteins as spacers. The implications for engineering organized multilayers of antibodies for optical biosensors are reviewed.