Immuno-atomic force microscopy characterization of adsorbed fibronectin.

The fibronectin (Fn) binding conformation on mica and ultraflat poly(D,L-lactide-co-glycolide) (UPLGA) was characterized using atomic force microscopy (AFM). AFM topographic images showed that Fn was in an extended form on mica and in a compact structure on UPLGA. With immuno-AFM, an antibody (Ab(hep)) was used to characterize the Fn binding conformation. When Fn opens its binding site for an antibody upon adsorption to a surface, the resulting Fn-antibody complex creates an additional peak in the sample's height distribution. Immuno-AFM uses this change to detect antigen-antibody binding. In this letter, height histograms (distributions) were generated using the mean true height of molecules, which was measured by examining the histogram for each individual molecule and subtracting the mica background. Mean true height values were obtained from the histograms and showed that Fn and Ab(hep) formed complexes on mica, signifying that one of the heparin binding sites on Fn was open when Fn was adsorbed to mica. The mean true height of the Fn-antibody complex from the histogram is greater than expected, suggesting that the antibody had pulled the extended "arms" of Fn together and caused an Fn conformation change upon binding. The height histograms can illustrate the Fn binding conformation and other antigen-antibody binding.