The orientation of a monoclonal, anti-streptavidin human IgG1 antibody on a model hydrophobic, CH(3)-terminated surface (1-dodecanethiol self-assembled monolayer on gold) was studied by monitoring the mechanical coupling between the adsorbed layer and the surface as well as the binding of molecular probes to the antibodies. In this study, the streptavidin antigen was used as a probe for the Fab portions of the antibody, while bacteria-derived Protein G' was used as a probe for the Fc region. Bovine serum albumin (BSA) acted as a blocking protein. Mono layer coverage occurred around 468 ng/cm(2). Below 100 ng/cm(2), antibodies were found to adsorb flat-on, tightly coupled to the surface and unable to capture their antigen, whereas the Pc region was able to bind Protein G'. At half-monolayer coverage, there was a transition in the mechanism of adsorption to allow for vertically oriented antibodies, as evidenced by the binding of both Protein G and streptavidin as well as looser mechanical coupling with the surface. Mono layer coverage was characterized by a reduced level in probe binding per antibody and an even less rigid coupling to the surface.