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

Science Bulletin, Dec 2011

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.

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

LI Mi 1 2 LIU LianQing 2 XI Ning 0 2 WANG YueChao 2 DONG ZaiLi 2 LI GuangYong 2 4 XIAO XiuBin 3 ZHANG WeiJing 3 0 Department of Electrical and Computer Engineering, Michigan State University , East Lansing 48824, USA 1 Graduate University of Chinese Academy of Sciences , Beijing 100049, China 2 State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences , Shenyang 110016, China 3 Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences , Beijing 100071, China 4 Department of Electrical and Computer Engineering, University of Pittsburgh , Pittsburgh 15261, USA 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. - The invention of atomic force microscopy (AFM) [1] allows researchers to investigate the topographical structure and specific molecular interactions between individual live cells and molecules, thereby revolutionizing the research methods used in the field of life sciences. The resolution of light microscopy is limited to the wavelength of the light source and, therefore, information on a nanometer scale is not accessible; also, electron microscopy requires vacuum conditions, which means that live samples cannot be examined using this technique [2]. Compared with light microscopy and electron microscopy, AFM has sub-nanometer resolution [3], can work under various conditions such as air, *Corresponding authors (email: ; ; ) liquid, and/or a vacuum, and allows the visualization of live cells and native biomolecules without the need for staining or fixation [4]. These advantages make AFM widely applicable to life sciences, a field in which research into individual cells/molecules using AFM is at the frontier [57]. As a result of significant advances over the last two decades, AFM has evolved into a multifunctional tool [4]. Chemical treatment of the AFM tip and substrate allows the measurement of specific protein-protein binding forces using the force curve mode, a technique known as singlemolecule force spectroscopy (SMFS) [8]. In SMFS, ligands/antibodies are linked to AFM tips and receptors/antigens are then bound to a substrate. Using the functionalized tip to obtain force curves for the protein-coated The Author(s) 2011. This article is published with open access at Springerlink.com substrate, the rupture forces between the antibodyantigen/receptor-ligand can be measured. SMFS was first used in the mid-1990s. Florin et al. [9] measured the rupture force for the biotin-avidin complex immobilized on biotinylated agarose beads by linking avidin to the AFM tip. Hinterdorfer et al. [10] measured the rupture force between human serum albumin (HSA) and anti-HSA by linking the anti-HSA onto an AFM tip using polyethylene glycol (PEG) linker molecules and linking HSA onto mica surfaces. In addition to measuring the molecular forces between protein-coated substrates, SMFS can also measure molecular forces at the cell surface. Puntheeranurak et al. [11] measured the specific binding forces between the Na+-glucose co-transporter (SGLT1) to its antibodies on Chinese hamster ovary (CHO) cells, and Shi et al. [12] investigated the interaction forces between Heregulin and HER3 on human embryonic kidney (HEK) 293 cells. The clinical application of Rituximab (a monoclonal antibody against CD20) during the last decade has highlighted a marked difference in efficacy in the treatment of patients with non-Hodgkins lymphoma [13]. The target of Rituximab is the CD20 antigen, which is a tetra-spanning membrane protein expressed on mature B cells and by most B cell lymphomas [14]. After binding to CD20 on the cell surface, Rituximab induces target cell lysis via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and programmed cell death (PCD) [15]. Rituximab achieved unprecedented success and, particularly in combinati (...truncated)


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Mi Li, LianQing Liu, Ning Xi, YueChao Wang, ZaiLi Dong, GuangYong Li, XiuBin Xiao, WeiJing Zhang. Detecting CD20-Rituximab interaction forces using AFM single-molecule force spectroscopy, Science Bulletin, 2011, pp. 3829-3835, Volume 56, Issue 35, DOI: 10.1007/s11434-011-4789-0