Detecting CD20-Rituximab interaction forces using AFM single-molecule force spectroscopy

The invention of atomic force microscopy (AFM) has provided new technology for measuring specific molecular interaction forces. Using AFM single-molecule force spectroscopy (SMFS) techniques, CD20-Rituximab rupture forces were measured on purified CD20 proteins, Raji cells, and lymphoma patient B cells. Rituximab molecules were linked onto AFM tips using AFM probe functionalization technology, and purified CD20 proteins were attached to mica using substrate functionalization technology. Raji cells (a lymphoma cell line) or lymphoma patient cells were immobilized on a glass substrate via electrostatic adsorption and chemical fixation. The topography of the purified CD20 proteins, Raji cells, and patient lymphoma cells was visualized using AFM imaging and the differences in the rupture forces were analyzed and measured. The results showed that the rupture forces between the CD20 proteins on Raji cells and Rituximab were markedly smaller than those for purified CD20 proteins and CD20 proteins on lymphoma patient B cells. These findings provide an effective experimental method for investigating the mechanisms underlying the variable efficacy of Rituximab.