Experimental
Single-molecule selection and recovery of structure-specific antibodies
using atomic force microscopy
Luda S. Shlyakhtenko, PhD,
a
Bin Yuan, PhD,
b
Sharareh Emadi, PhD,
b
Yuri L. Lyubchenko, PhD, DSc,
a
Michael R. Sierks, PhD
b,
⁎
a
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska, USA
b
Department of Chemical Engineering, Arizona State University, Tempe, Arizona, USA
Received 5 March 2007; accepted 22 June 2007
Abstract Protein misfolding and aggregation are a common thread in numerous diseases including
Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, diabetes, and prion-related
diseases. Elucidation of the role played by the various protein forms in these diseases requires
reagents that can target specific protein forms. Here we present a method to isolate antibodies that
bind to a specific protein form. We combined the imaging and nanomanipulation capabilities of
atomic force microscopy (AFM) with the protein diversity of phage display antibody libraries to
develop a technology that allows us to recover a single antibody molecule that is bound to a single
protein molecular target. The target protein-antibody complex is first imaged by AFM, the AFM tip
is then manipulated by nanolithography over the target antibody to recover the associated phage, and
the antibody gene is recovered from the single phage particle by polymerase chain reaction.
© 2007 Published by Elsevier Inc.
Key words: Phage display; Single-chain antibody fragment; Atomic force microscopy; Single molecule; Protein misfolding
Antibodies have tremendous potential as therapeutics and
diagnostics. Nearly a third of new biopharmaceuticals are
recombinant antibodies [1], and monoclonal antibodies are a
standard for diagnostic protocols [2,3]. Nanosensor technol-
ogy developments now permit the detection of target
antigens at extremely low concentrations [4] and in in vivo
applications [5]. If suitable antibody reagents were available,
such technology could be used to detect the presence of
misfolded or mutant proteins or cells in vivo. Here we
describe a single-molecule selection technique that combines
the diversity of phage display technology [6] with the
imaging capabilities of atomic force microscopy (AFM),
allowing us to simultaneously visualize individual protein
morphologies and recover an antibody that binds a
specifically selected target. The ability to detect the presence
of misfolded or mutant proteins, altered DNA, or cancerous
cells in vivo can have tremendous therapeutic value in early
diagnosis of a host of different diseases.
Protein misfolding, aggregation, and deposition is a
common thread in numerous diseases including neurode-
generative disorders such as Alzheimer's, Parkinson's, and
Huntington's diseases, Down syndrome, systemic and
localized amyloidoses, and transmissible encephalopathies
(see examples [7-13]). Numerous protein misfolding dis-
eases form similar aggregate morphologies including large
fibril, spherical protofibril, and smaller aggregate or
oligomer structures. Although all of these structures occur,
their respective role in the progression of protein deposition
diseases is not known, however, recent studies [14] have
shown that the oligomeric intermediate morphologies are
cytotoxic regardless of protein sequence. Even though the
proteins involved in theses diseases vary greatly in sequence
and length, they can all generate morphologically similar
structures that are recognized by the same antibodies [15].To
Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 192 – 197
www.nanomedjournal.com
No conflict of interest was reported by the authors of this paper.
⁎
Corresponding author. Department of Chemical Engineering, Arizona
State University, Box 876006, Tempe, AZ 85287-6006, USA.
E-mail address: sierks@asu.edu (M.R. Sierks).
1549-9634/$ – see front matter © 2007 Published by Elsevier Inc.
doi:10.1016/j.nano.2007.06.001