Interfacial investigations of antibody immobilization strategies

The study of protein adsorption onto solid surfaces has become an important area of study for the development of biocompatible materials. Atomic force microscopy (AFM) has been used to image adsorbed biomolecules with remarkable resolution and to measure the forces between protein-functionalized AFM tips and surfaces. Modified AFM tips and substrates have been used to investigate highly specific, discrete interactions between different biomolecules and may be used to map the orientation and conformation of surface adsorbed proteins. The orientation of molecules immobilized on an AFM probe and/or changes in the conformation of surface adsorbed proteins may influence the number of binding events and measured forces.; The influence of antibody immobilization strategy was evaluated with whole and fragmented antibodies. IgG was immobilized to amine-modified surfaces either in a specific orientation through the use of Protein A, or randomly cross-linked to the surface via glutaraldehyde to evaluate the influence of antibody orientation on antigen binding. To investigate the influence of tether length on antigen binding, surfaces were coated with gold and modified with different length bifunctional linkers containing a sulfhydryl reactive group that was used to covalently attach F(ab') fragments through the native thiol group in the protein. X-ray photoelectron spectroscopy and surface plasmon resonance (SPR) were employed to confirm the addition of the linkers and antibody immobilization to the gold surfaces. Oriented linker immobilized antibody F(ab') fragments were compared to F(ab') fragments immobilized directly onto a gold surface through the native sulfhydryl group on the protein. The influence of immobilization strategy on the antigen binding activity of immobilized antibodies was assessed using enzyme-linked immunosorbent assay (ELISA), quartz crystal microbalance (QCM), SPR and AFM adhesion force measurements.; These studies indicate that oriented antibody coated surfaces have higher antigen binding activities than surfaces prepared with random immobilization strategies. The influence of tether length was also found to be significant for solution binding assays and AFM interaction force measurements. Finally, the use of non-ionic surfactants was found to improve selectivity in AFM force measurement experiments by reducing non-specific protein-protein interactions that interfere with the detection of specific antibody-antigen binding events.