Immunosensing based on site-directed immobilization of antibody fragments and polymers that reduce nonspecific binding.

Antibody Fab'-fragments can be directly coupled onto gold, and the space between the fragments can be filled with protein repellent disulfide bearing polymers. Coupling of the antibody Fab'-fragments, and thus both the amount of nonspecific binding and antigen binding but also the ability to regenerate the layer, is dependent on the immobilization procedure. First, the immobilization has taken place by coupling the Fab'-fragments to the surface and thereafter attaching the polymer in the remaining space between the antibodies. Second, the Fab'-fragments have been added after the surface has been coated by polymer. Third, the Fab'-fragments and polymer have been added onto the surface from the same solution. Up to 80% of the antigen could be removed during regeneration, if proper concentrations of polymer and Fab'-fragments were immobilized onto the gold surface. Only about 60% of the antigen could be removed, when the fragments were coupled directly onto a clean Au surface before the polymer or if low concentrations of polymer were attached onto gold before the Fab'-fragments. The first immobilization method, however, showed the highest response to antigen.