Imaging recognition events between human IgG and rat anti-human IgG by atomic force microscopy

Abstract

Chemically immobilized rat anti-human immunoglobulin (IgG) monolayers on thiols modified gold substrates were fabricated using self-assembled monolayer (SAM) method. The antibody monolayers were imaged before and after free human IgG treated, whilst recognition events between antigen and antibody were monitored by contact mode atomic force microscopy (CM-AFM) and tapping mode AFM (TM-AFM), with topographic and/or phase images being recorded. The obtained images with different surface compositions show distinct nanostructures, indicating occurrence of recognition and binding events of antigen–antibody. The size of the observed surface structures of the antibody monolayer, when tip broaden effect had been taken into account, was very close to the actual size of the antibody molecule. Thus, these results suggest CM-AFM is capable of, and proven satisfactory in detecting protein–protein interactions (PPIs), providing the sample was prepared appropriately and the scanning parameters were set adequately. Moreover, phase imaging can serve as a real time contrast enhancement technique to TM-AFM in terms of highlighting edges and clearly observing fine features.