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.

Repelling and ordering: the influence of poly(ethylene glycol) on protein adsorption

“…Naturally, also in the case of biosensors, having access to specific binding sites of the protein is desirable. 26 In this work we study the influence of PEG concentration in mixed 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy-(polyethylene glycol)-2000] (DMPE-PEG2000) phospholipid monolayers on both the amount and the orientational ordering extent of the adsorbed proteins. We have chosen bovine serum albumin (BSA) and human fibrinogen (Fbg) as model systems to track the impact of PEG density on the order of large proteins.…”

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG

“…The functionalized surface showed a peculiar distribution and assembly of the protein A‐antibody complex. Furthermore, a symmetrical sectional profile was found to be representative of a unique antibody orientation (Welch, Scoble, Muir, & Pigram, ).…”

Impedance Detection of 3‐Phenoxybenzoic Acid Comparing Wholes Antibodies and Antibody Fragments for Biomolecular Recognition

“…However, the charge transfer resistance (7-15 kW-cm 2 ) of the sensor interface is reduced by more than one order of magnitude relative to that obtained (130-200 kW-cm 2 ) for amide bond formation. This reduces detection limits by several orders of magnitude, to 1.26 3 10 À8 M for subsequent site blocking by BSA adsorption and to 1.01 3 10 À8 M for subsequent backfilling with 6-mercapto-1-hexanol (MCH). Surprisingly, the detection limits for these two cases are almost identical, so MCH backfilling provides little benefit over BSA site blocking.…”

“…The sensitivity and selectivity of antibodybased biosensors depend in a complex manner on the surface density and orientation of biomolecules, and the extent to which their native conformation and flexibility are retained [1][2][3]. Although many species can be used for biomolecular recognition, antibody-based methods are most popular, where the antibody or antibody fragments are immobilized onto a solid surface.…”

“…Although many species can be used for biomolecular recognition, antibody-based methods are most popular, where the antibody or antibody fragments are immobilized onto a solid surface. The sensitivity and selectivity of antibodybased biosensors depend in a complex manner on the surface density and orientation of biomolecules, and the extent to which their native conformation and flexibility are retained [1][2][3]. The use of antibody fragments has been tested for several different biosensor technologies to improve biomolecular orientation, reduce biomolecular film thickness, and to develop alternative biomolecular immobilization methods [1][2][3].…”