Simultaneous Height and Adhesion Imaging of Antibody-Antigen Interactions by Atomic Force Microscopy

Abstract

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

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

“…Since lateral movement of the AFM tip can be done in a retracted position in force volume mode, lateral forces on the sample during scanning can be avoided. Image resolutions comparable to tapping mode AFM can be achieved [1159]. A fundamental problem in force volume mode is the relatively long acquisition time that can easily amount to tens of minutes and can give rise to excessive drift.…”

“…The ability to resolve specific interactions as antibody-antigen binding between AFM tip and sample surface with nanometer resolution by force distance curves [1188] makes the force volume mode an ideal tool for ''affinity imaging''. It has been applied to biological model systems as biotin-streptavidin [1189], intercellular adhesion molecule-1 (ICAM-1) and anti-ICAM-1 [1159], ferritin-anti-ferritin [1190], fibrinogen-anti-fibrinogen [1191] and tymine and adenine [1192,1193]. The obvious potential for mapping distributions of biomolecules on cell surfaces has been exploited to image distribution of mannan polymers on the yeast cells [1194], sugar chains on tissue sections of the rat vomeronasal epithelium [1195], receptor-associated protein binding proteins on 3T3 fibroblasts [1196], vitronectin receptors on a murine osteoblastic cell [1197], vascular endothelial growth factor (VEGF) receptor on bovine aortic endothelial cells [1198], tyrosine kinase A on PC 12 nerve cells [1199], calcitonin receptors on bone cells [1200].…”

Combining constitutive materials modeling with atomic force microscopy to understand the mechanical properties of living cells

“…More recent developments in AFM now allow the detection of molecular recognition events between single molecules using ligands attached to AFM tips for the recognition of receptors bound on rigid surfaces (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). By monitoring the cantilever def lection during approach-retraction cycles (i.e., forcevolume͞force-distance curves) at a constant (lateral) position on the sample, unbinding forces (i.e., the maximum force at the moment of receptor-ligand detachment) have been determined for various ligand-receptor pairs, including biotin-avidin (13,14,21), DNA bases (15), antibody-antigen (16)(17)(18)(19)(20)(21)(22), and cellrecognition proteins (23).…”

Interactions of the streptococcal C5a peptidase with human fibronectin

“…The NHS-PEG-PDP cross-linker [33] was chosen because the PDP group will bind to the cystine on the GST tag and the NHS group will bind to an amine on the tip surface. The effectiveness of this cross-linker has been proven in other single molecule studies [32,34,35].…”