Feasibility of measuring antigen–antibody interaction forces using a scanning force microscope

Abstract

The molecular affinity scanning force microscopy (MASFM) described in this study was developed in an effort to test the possibility of antigen-antibody binding measurement using forceseparation distance profiles. The MASFM configuration was comprised of a spherical glass bead as an MASFM probe, to which the fluorescein antigen has been covalently attached, and a silicon dioxide-based substrate, to which the antifluorescyl IgG antibody was covalently bound. The bead was glued to the tip of a commercial SFM cantilever. Adhesion forces have been measured between two different specific antigen-antibody pairs and between nonspecific surfaces bearing only glycidoxypropylsilane immobilization chemistry. In force-separation (F-s) measurements, nonspecific forces displayed relatively few force discontinuities and mean adhesion forces lower than those found for specific antigen-antibody measurements. Force-separation profiles measured between specific antigen-antibody pairs showed many discontinuities and had higher mean forces. Positive controls revealed that the mean forces were slightly reduced by the addition of free ligand. The magnitude of mean forces did not correlate with the respective activation enthalpies of the proteins, as would be expected. At lower force values the force histograms for the specific pairs and for positive controls were indistinguishable. None of the force-separation data sets could fit a Poisson discrete-force model. This statistical analysis showed a large relative contribution from nonspecific interactions. It is concluded that the use of the large sphere as an SFM probe is counterproductive: while the large sphere does sample a larger number of specific interactions during each measurement, it also samples at the same time a large proportion of nonspecific forces. The presence of the nonspecific force contributions is likely due to the deformation of the polymerized GPS spacer layer which is thought to be delaminated from the surface upon the application of tension across the specific antigen-antibody bonds.

Direct measurement of interactions between adsorbed vitronectin layers: The influence of ionic strength and pH

“…This force separation profile is similar to interaction-force curves observed in polymer systems and has also been reported for protein systems 26,32 and by groups investigating molecular recognition events. 24,51 The larger separation distance than expected for the stretched protein molecule may be a result of molecular aggregation between the adsorbed proteins and bridging between the surfaces.…”

“…20 In addition, the AFM allows samples to be investigated in aqueous environments, which is important for biological samples, where they can be examined in vitro with high resolution and without drying or using fixative material. 21,22 Direct interaction force measurements have been successfully developed in recent years to study adsorbed protein layers, for example, measurement of single molecule forces between specific protein pairs, 23,24 or antigen/antibody pairs, [25][26][27] and of interaction forces with a physicochemical nature. 28 -32 The adsorption of proteins is strongly influenced by their stability in solution.…”

Reflection Interference Contrast Microscopy Combined with Scanning Force Microscopy Verifies the Nature of Protein-Ligand Interaction Force Measurements

“…This combination was used to study interaction forces and distances between receptor/ligand pairs. The example system presented here uses antifluorescyl IgG/ fluorescein pairs covalently immobilized to glass or quartz substrates with a glycidoxypropylsilane (GPS) linker (Stuart, 1997). The fluorescein ligand was immobilized to a glass sphere, which was subsequently glued to the apex of a commercial SFM cantilever.…”

“…Glass coverslips (1Љ round) and glass beads (5-50 m) were prepared as detailed by Stuart (1997) and Stuart and Hlady (1998). Briefly, the substrates were covalently derivatized with 3-glycidoxypropyltrimethoxysilane (GPS) (Hüls/United Chemical Technologies, Bristol, PA) to create an amine-reactive surface film.…”

“…Both substrate and probe were thoroughly characterized to establish the effectiveness of the surface chemistry (Stuart, 1997). Contact angle measurements monitored changes in the surface energy of each layer, and x-ray photoelectron spectroscopy quantified the elemental compositions.…”