![]() 15 Covalent attachment of the antibody, via its functional groups, to chemically engineered substrates has resulted in further improvements in antibody density, though often these methods are not site-directed and unfavorable random orientation can occur. 12–14 The substrate design has increased the capabilities of immunoassays by improving antibody binding capacity and reducing denaturation at the surface. 11 While this method offers the simplest attachment pathway, it is uncontrollable, and antibodies can be immobilized in a randomly oriented manner, denatured, or displaced in later steps by washing. Physical adsorption of antibodies onto traditional immunoassay solid supports, such as polystyrene, occurs via hydrophobic and electrostatic interactions. Future perspectives and recommendations are offered in conclusion. 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. 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). ![]() ![]() The introduction describes the importance of oriented antibodies for maximizing biosensor capabilities. This review describes the most recent methods for oriented antibody immobilization and the characterization techniques employed for investigation of the antibody state. 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. Orientation of surface immobilized capture proteins, such as antibodies, plays a critical role in the performance of immunoassays.
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