Detection and localization of individual antibody-antigen recognition events by atomic force microscopy

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands, For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN, Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule, The two Fab fragments of the antibody were found to bind independently and with equal probability, The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms, This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm, It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.