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.
Highlights
Immunodiagnostics, protein biochips, and biosensors employed for antigen detection and quantification from biological samples often employ recognition proteins such as antibodies.[1,2,3,4] Assay sensitivity is dependent on immobilization of the capture antibodies onto a solid support with a sufficient surface density, a conformation that is representative of their native, solution-phase state, and an orientation that maximizes their antigen capture potential.Generally, hydrophobic amino acids are internalized in a correctly folded protein structure, leaving hydrophilic residues at the antibody surface, with chemically reactive functionalities, including amine, carboxyl, and hydroxyl groups
This review describes the most recent methods for oriented antibody immobilization and the characterization techniques employed for investigation of the antibody state
Plasma polymers have been produced from a diverse range of monomers, including allylamine,[25,26] cyclopropylamine,[27] bromine,[28] polyethylene glycol (PEG), diethylene glycol dimethyl ether,[29,30,31] and many others,[32,33] providing a broad spectrum of chemical functionalities for subsequent protein grafting steps, including the option for patterning.[34,35,36]
Summary
Immunodiagnostics, protein biochips, and biosensors employed for antigen detection and quantification from biological samples often employ recognition proteins such as antibodies.[1,2,3,4] Assay sensitivity is dependent on immobilization of the capture antibodies onto a solid support with a sufficient surface density, a conformation that is representative of their native, solution-phase state, and an orientation that maximizes their antigen capture potential. Hydrophobic amino acids are internalized in a correctly folded protein structure, leaving hydrophilic residues at the antibody surface, with chemically reactive functionalities, including amine, carboxyl, and hydroxyl groups. Disulfides, such as those that contribute to the hinge region, can be reduced to make thiol species that can be conjugated. Improvements in antibody density, though often these methods are not site-directed and unfavorable random orientation can occur.[16] In an ideal scenario, antibodies should be immobilized in their native form, without the need for introduced functional groups, in a homogeneous arrangement such that their antigen binding sites are free from steric hindrance and are oriented so as to maximize complementary binding. The advantages and shortfalls of strategies and techniques will be addressed, and the review will conclude with future perspectives and recommendations
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