USE OF SCANNING PROBE MICROSCOPY AND SURFACE-PLASMON RESONANCE AS ANALYTICAL TOOLS IN THE STUDY OF ANTIBODY-COATED MICROTITER WELLS

Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) have been employed to study microtiter wells used for enzyme linked im munosorbant assays of the protein ferritin. Two methods of immobilizat ion of a ferritin antibody on microtiter well surfaces were evaluated. Namely, passive adsorption of unmodified antibody, and specific linki ng of biotinylated antibody via a streptavidin-coated microtiter well surface. Scanning probe microscopy (SPM) data clearly show films and n etworks of monoclonal antibody on the well surfaces and individual fer ritin molecules bound by the antibody. SPM images give an average mole cular diameter for the ferritin bound to the antibody complex of 14.8 +/- 1.0 nm for the STM data and approximately 30 nm for the AFM data. SPM analysis shows that only 5% of total antibody passively adsorbed o n the well surface is functional compared with the 60% on biotinylated antibody on the streptavidin surface. In similar experiments using su rface plasmon resonance (SPR) experimental data also indicated an incr ease in the functionality of a streptavidin-coated surface compared wi th a blank surface. The results highlight the correlation between STM and AFM data and the use of SPR as a correlative technique to SPM for the study of biomaterial surface interactions.