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.