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.
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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)