Abstract

The purpose of this study is to investigate conformations of antibodies immobilized on three kinds of solid surfaces modified with APTES, AEAPTES or GA and their antigen-binding affinities. In the recent developments in biosensors, although the geometric structure of antibodies determines their sensitivity, few studies have focused on their conformations and its effect on their antigen-binding capacity due to a lack of applicable analysis techniques. In this work, specular and off-specular X-ray reflectivity measurement were employed, which enables us to analyze vertical and lateral multilayer structure. The results showed that the antigen-binding affinity on GA was highest among antibodies immobilized on three modified surfaces, although their conformations were almost the same as 'flat-on' analyzed by specular X-ray reflectivity. In contrast, lateral layer structure analysis by off-specular reflection revealed that protein layer on GA before antigen-binding had a relatively-long lateral correlation length and a low fractal dimension which would cause the reduction of steric hindrance for antigens' access to binding sites of antibodies.

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