A method for immobilization of ligand antibody to improve the efficiency and sensitivity of a sandwich enzyme-linked immunosorbent assay (ELISA) was investigated by the use of anti-TNF-alpha monoclonal antibody chemically conjugated with a polystyrene-binding peptide (PS-tag) and an intelligent microtiter plate with large surface area. We compared both adsorption and antigen-binding activity of the ligand antibody (mAb) and mAb with the PS-tag (mAb-PS-tag) on 3 different PS plates: a hydrophobic PS plate (PS-F-1 plate), a hydrophilic PS plate (PS-A plate), and an intelligent microtiter plate packed with PS beads (PS-E plate). Contact areas of the PS-E plate toward ligand antibody solutions were 7-fold larger than those of conventional PS-F-1 and PS-A plates and consequently, both mAb and mAb-PS-tag were efficiently immobilized on the surface of the PS-E plate due to the significantly enhanced surface area. In particular, when the non-ionic surfactant, Tween20, was present during adsorption of the ligand antibodies, the mAb-PS-tag was site-specifically immobilized on the surface of the PS-E plates and exhibited the highest specific antigen-binding activity. High specific antigen-binding activities were preserved, even though the density of the mAb-PS-tag immobilized on the PS-E plate was reduced with application of smaller volumes. Consequently, the decline in the signal intensities detected from the sandwich ELISA on the PS-E plate was negligible, even when one-fourth the original volume of mAb-PS-tag solution was used to coat the plate. The ligand antibody was immobilized with retention of high antigen-binding activity due to the large surface area of the PS-E plate, specific recognition of the PS-tag, and minimal non-specific interaction of the ligand antibody due to use of Tween 20. Thus, the immobilization method developed in this study resulted in an efficient and highly sensitive sandwich ELISA system that requires smaller amounts of ligand antibodies. This method may be useful for fabrication of protein-based biochips, such as antibody chips.