Influence of Surfactants and Antibody Immobilization Strategy on Reducing Nonspecific Protein Interactions for Molecular Recognition Force Microscopy

Abstract

Specific and nonspecific interactions between antibody-modified probes and substrate-immobilized proteins were monitored by atomic force microscopy (AFM). Probes were modified with anti-ovalbumin IgG antibodies immobilized in either an oriented or a random manner. The oriented immobilization of whole IgG was accomplished through the use of Protein A, and random immobilization was carried out with glutaraldehyde. Nonspecific interactions may lead to false detection of antibody-antigen binding events even when the antigen binding sites are properly positioned by an oriented immobilization strategy. Thus, nonionic and zwitterionic surfactants, including Tween 20, Tween 80, Triton X-100, and CHAPS, were evaluated to determine if nonspecific binding events could be reduced without compromising the desired specific antibody-antigen binding. Enzyme-linked immunosorbent assay and surface plasmon resonance assays were also employed to study antibody-antigen binding as a function of immobilization strategy and surfactant concentration. The data from these studies indicate that Protein A can be used to immobilize whole IgG onto AFM probes for force measurement experiments and that a surfactant is useful for improving the selectivity for such measurements.

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…In order to avoid this problem, one can perform the measurements in a particular liquid with small van der Waals force or attach one or both of the interacting molecules to the tip and/or to the sample via a spacer, i.e., a few nanometers long molecule which is stretched during the detachment and makes the rupture of the bond occur at distances greater than the jump-off-contact distance. Also, particular techniques of immobilization of the interacting molecules and the use of particular surfactants can reduce the non-specific interactions [1117].…”

“…Using ligands which bind to a defined region of the adsorbed protein, force-displacement curves are acquired, and the quantity of formed complexes gives an estimate of the amount of protein oriented in such a way as to allow ligand binding. (See also [1117,1141]. ) Gutsmann et al [1142] have used short cantilevers (length smaller than 20 mm) to improve the force resolution below 10 pN range.…”

A label-free immunosensor array using single-chain antibody fragments

“…All measurements were carried out in the liquid cell at 18°C Ϯ 0.1°C, while the cantilever microarray was immersed in HBS buffer. As a surfactant Tween 20 was added to the buffer to block nonspecific protein-protein interactions (19). Before the binding assay, all eight cantilevers in the array were calibrated to evaluate their mechanical homogeneity (see Materials and Methods).…”

“…Thus, the sensitivity of surface plasmon resonance (SPR) and quartz crystal microbalance sensors was significantly improved by using antibody fragments (13,19), which can be bound covalently to the sensor surface in an oriented manner by using their C-terminal SH groups.…”

Grafted-double walled carbon nanotubes as electrochemical platforms for immobilization of antibodies using a metallic-complex chelating polymer: Application to the determination of adiponectin cytokine in serum

“…After functionalization of DWCNTs (step 1), 3 mL of a 0.5 mg HOOC-Phe-DWCNTs per mL aqueous dispersion which also contained 15 mg/mL Tween 20 ™ were casted on the SPCE surface and dried under IR radiation. Tween 20 acted as a suspension stabilizer and its role in reducing nonspecific protein adsorption via primarily hydrophobic interactions has been also reported (Brogan et al, 2004). Then, 3 mL of Mix&Go polymer solution were dropped onto the HOOC-Phe-DWCNTs/SPCE and left standing for 1 h at room temperature.…”