Site-directed immobilization of antibody onto solid surfaces for the construction of immunochip

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity, i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidin-coated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.