title: Influence of surfactants and antibody immobilization strategy on reducing nonspecific protein interactions for molecular recognition force microscopy.
abstract: Specific and nonspecific interactions between antibody-modified probes and substrate-immobilized proteins were monitored by atomic force microscopy (AFM). Probes were modified with anti-ovalbumin IgG antibodies immobilized in either an oriented or a random manner. The oriented immobilization of whole IgG was accomplished through the use of Protein A, and random immobilization was carried out with glutaraldehyde. Nonspecific interactions may lead to false detection of antibody-antigen binding events even when the antigen binding sites are properly positioned by an oriented immobilization strategy. Thus, nonionic and zwitterionic surfactants, including Tween 20, Tween 80, Triton X-100, and CHAPS, were evaluated to determine if nonspecific binding events could be reduced without compromising the desired specific antibody-antigen binding. Enzyme-linked immunosorbent assay and surface plasmon resonance assays were also employed to study antibody-antigen binding as a function of immobilization strategy and surfactant concentration. The data from these studies indicate that Protein A can be used to immobilize whole IgG onto AFM probes for force measurement experiments and that a surfactant is useful for improving the selectivity for such measurements.
authors.affiliation: Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Brogan
authors.forename: Jae Ho
authors.initials: JH
authors.lastname: Shin
authors.forename: Mark H
authors.initials: MH
authors.lastname: Schoenfisch
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D018625:Microscopy, Atomic Force / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D000906:Antibodies; D011506:Proteins; D013501:Surface-Active Agents