Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay

Abstract

Antibody immobilization strategies (random, covalent, orientated and combinations of each) were examined to determine their performance in a surface plasmon resonance-based immunoassay using human fetuin A (HFA) as the model antigen system. The random antibody immobilization strategy selected was based on passive adsorption of anti-HFA antibody on 3-aminopropyltriethoxysilane (APTES)-functionalized gold (Au) chips. The covalent strategy employed covalent crosslinking of anti-HFA antibody on APTES-functionalized chips using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (SNHS). The orientation strategy used passive adsorption of protein A (PrA) on Au chips, with subsequent binding of the anti-HFA antibody in an orientated fashion via its fragment crystallisable (Fc) region. In the covalent-orientated strategy, PrA was first bound covalently, to the surface, which in turn, then binds the anti-HFA antibody in an orientated manner. Finally, in the most widely used strategy, covalent binding of anti-HFA antibody to carboxymethyldextran (CM5-dextran) was employed. This immobilization strategy gave the highest anti-HFA antibody immobilization density, whereas the highest HFA response was obtained with the covalent-orientated immobilization strategy. Therefore, the covalent-orientated strategy was the best for SPR-based HFA immunoassay and can detect 0.6-20.0 ng/mL of HFA in less than 10 min.

How Antibody Surface Coverage on Nanoparticles Determines the Activity and Kinetics of Antigen Capturing for Biosensing

“…We attribute these low values of active antibodies at equilibrium to the orientation, denaturation, and/or steric availability of the antibodies. The EDC−sNHS chemistry couples the antibodies at a random orientation, 22,23 including immobilization via the NH 2 groups close to the F ab fragment, 11,13 which can decrease the ability to capture cTnI from solution. Second, the antibody activity may be lowered by unfolding and spreading of antibodies on the nanoparticle surface.…”

Immobilization of Antibodies and Enzymes on 3-Aminopropyltriethoxysilane-Functionalized Bioanalytical Platforms for Biosensors and Diagnostics

“…As an example, Protein A covalently bound on a silanized Au-coated SPR chip by EDC-SNHS can be useful for the oriented immobilization of antihuman fetuin A (anti-HFA) Ab. 63 This orientation strategy results in a superior analytical performance as compared to covalent, noncovalent, Protein A noncovalently bound, and commercial CM5 (carboxymethyl dextran) SPR chip-based Ab immobilization strategies. Although these intermediate proteins have been widely used for direct binding assays, such as those based on SPR, microcantilevers, and quartz crystal microbalance (QCM), they are not widely employed for sandwich IAs.…”

“…61 Recently, this procedure has been proven in SPRbased immunoassays (IAs) using Au-coated SPR chips. 62,63 The Au SPR chip is simply treated with a piranha solution for 2 min to generate the desired surface hydroxyl groups and incubated in 2% (v/v) APTES in water for 1 h. A typical mixture of the piranha solution is 3:1 (v/v) concentrated sulfuric acid to 30% H 2 O 2 solution.…”

Orientation and characterization of immobilized antibodies for improved immunoassays (Review)

“…Vashist et al 140 utilized SPR as an assay method to investigate antibody immobilization strategies; random, covalent (EDC/NHS), oriented with adsorbed Protein A followed by antibody binding, covalent-oriented Protein A followed by antibody binding, and last covalent-CM5-dextran binding. SPR determined that the mass of antibody immobilized was greatest for the CM5 system.…”