The enhanced analytical performances of immunoassays that employed site-directly immobilized antibodies as the capture binders have been functionally characterized in terms of antigen-antibody complex formation on solid surfaces. Three antibody species specific to cardiac troponin I, immunoglobulin G (IgG), Fab, and F(ab')(2) were site-directly biotinylated within the hinge region and then immobilized via a streptavidin-biotin linkage. The new binders were more efficient capture antibodies in the immunoassays compared to randomly bound IgG, particularly, in the low antibody density range. The observed improvements could have resulted from controlled molecular orientation and also from flexibility, offering conditions suitable for binding complex formations.