A comparative study on antibody immobilization strategies onto solid surface

Abstract

Antibody immobilization onto solid surface has been studied extensively for a number of applications including immunoassays, biosensors, and affinity chromatography. For most applications, a critical consideration regarding immobilization of antibody is orientation of its antigen-binding site with respect to the surface. We compared two oriented antibody immobilization strategies which utilize thiolated-protein A/G and thiolated-secondary antibody as linker molecules with the case of direct surface immobilization of thiol-conjugated target antibody. Antibody immobilization degree and surface topography were evaluated by surface plasmon resonance and atomic force microscope, respectively. Protein A/G-mediated immobilization strategy showed the best result and secondary antibody-mediated immobilization was the worst for the total immobilization levels of target antibodies. However, when considering real-to-ideal ratio for antigen binding, total target antigen binding levels (oriented target antibody immobilization levels) had the following order: secondary antibody-mediated immobilization>protein A/G-mediated immobilization>direct thiol-conjugated immobilization. Thus, we confirmed that protein A/G- and secondary antibody-mediated strategies, which consider orientation of target antibody immobilization, showed significantly high antigen binding efficiencies compared to direct random immobilization method. Collectively, the oriented antibody immobilization methods using linker materials could be useful in diverse antibody-antigen interaction-involved application fields.