Force measurement of specific antibody-antigen interactions in pH-varied liquid environments

The study reports the atomic force microscopy to measure the unbinding force of single antibody-antigen pair. The force measurements were conducted with the tip functionalized with antigens to its corresponding contact surface of the glass substrate functionalized with antibodies in aqueous solutions. In the approach and retraction of the AFM operation procedure, the contact force is greatly related, which occurs in retraction step. The contact forces are involved regular adhesion, nonspecific and specific bindings. Of great importance is the protein specific binding that demonstrates the shift of a slope in retraction step in apparent contrast to the nonspecific binding. The shifted sharp pull-off curve is attributed to the prior elongation of protein pair under external stretching and subsequent rupture of their bindings, the greatest unbinding forces were found in a range of nearly pH 7, indicating the value of 256.4 ± 48.9 pH at pulling velocity of 166.7 nm/s. A sharp decrease of the unbinding force occurs below pH 6.7, and a gradual decrease of environment found beyond pH 8.0. This is the first time that single human IgG1 and anti-human IgG1 pair interaction was quantitatively measured under the liquid environment from pH 2.0 to pH 10.0. The results are significant and provide direct unbinding force evidences in nearly realistic environments.