title: Orientation and characterization of immobilized antibodies for improved immunoassays (Review).
abstract: Orientation of surface immobilized capture proteins, such as antibodies, plays a critical role in the performance of immunoassays. The sensitivity of immunodiagnostic procedures is dependent on presentation of the antibody, with optimum performance requiring the antigen binding sites be directed toward the solution phase. This review describes the most recent methods for oriented antibody immobilization and the characterization techniques employed for investigation of the antibody state. The introduction describes the importance of oriented antibodies for maximizing biosensor capabilities. Methods for improving antibody binding are discussed, including surface modification and design (with sections on surface treatments, three-dimensional substrates, self-assembled monolayers, and molecular imprinting), covalent attachment (including targeting amine, carboxyl, thiol and carbohydrates, as well as "click" chemistries), and (bio)affinity techniques (with sections on material binding peptides, biotin-streptavidin interaction, DNA directed immobilization, Protein A and G, Fc binding peptides, aptamers, and metal affinity). Characterization techniques for investigating antibody orientation are discussed, including x-ray photoelectron spectroscopy, spectroscopic ellipsometry, dual polarization interferometry, neutron reflectometry, atomic force microscopy, and time-of-flight secondary-ion mass spectrometry. Future perspectives and recommendations are offered in conclusion.
authors.affiliation: Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, Melbourne, VIC 3086, Australia and CSIRO Manufacturing, Clayton, VIC 3168, Australia.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Welch
authors.affiliation: CSIRO Manufacturing, Clayton, VIC 3168, Australia.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Scoble
authors.affiliation: CSIRO Manufacturing, Clayton, VIC 3168, Australia.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Muir
authors.affiliation: Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, Melbourne, VIC 3086, Australia.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Pigram
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology; D057930:Click Chemistry; D004247:DNA / Q000737:chemistry; D007118:Immunoassay / Q000379:methods*; D018625:Microscopy, Atomic Force; D054802:Molecular Imprinting; D010455:Peptides / Q000737:chemistry; D056951:Photoelectron Spectroscopy; D011108:Polymers / Q000737:chemistry
chemical_list: D000906:Antibodies; D055910:Antibodies, Immobilized; D010455:Peptides; D011108:Polymers; D004247:DNA