3D antibody immobilization on a planar matrix surface

The amount of active capture antibodies immobilized per unit square is crucial to developing effective antibody chips, biosensors, immunoassays and other molecular recognition technologies. In this study, we present a novel yet simple method for oriented antibody immobilization at high density based on the formation of an orderly, organized aggregation of immunoglobulin G (IgG) and staphylococcal protein A (SPA). Following the chelation of His-tag with Ni2+, antibodies were immobilized on a solid surface in a three-dimensional (3D) manner and exposed the analyte receptor sites well, which resulted in a substantial enhancement of the analytical signal with more than 64-fold increase in detection sensitivity. Pull-down assays confirmed that IgG antibody can only bind to Ni2+ matrix indirectly via a SPA linkage, where the His-tag is responsible for binding Ni2+ and homologous domains are responsible for binding IgG Fc. The immobilization approach proposed here may be an attractive strategy for the construction of high performance antibody arrays and biosensors as long as the antibody probe is of mammalian IgG.