Biomedical Engineering Reference
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values of (10
−
3
s
−
1
10
2
s
−
1
) put into evidence the presence of a slow and a
fast processes; with the switching between the two processes being evident from the
abrupt change in the unbinding force plot when higher loading rates were applied.
The
k
off
value corresponding to the slow process agrees with that measured by SPR
(3
.
·
and 1
5
10
−
3
s
−
1
). Accordingly, they deduced that the DFS data could provide a good
description of the zero-force SPR measurements when they are taken at very low
loading rate and this should be kept in mind when comparing DFS and SPR data.
Sulchek et al. have used DFS to study the mucin 1 peptide in interaction with
a recombinant antibody selected through a library screening (Sulchek et al., 2005).
Mucin 1 is a transmembrane glycoprotein expressed in a variety of epithelial tissues,
and it is involved in the adhesion process to neighboring cells. Since mucin 1 has
been found to be overexpressed in some cancers, it may represent a suitable target
for a family of immunotherapeutics for cancer treatment. Searching for information
at single molecule level has been therefore conceived in the perspective of designing
more effective drugs. A particular attention has been devoted to reliably discriminate
between single and multiple unbinding events of mucin 1-involving complexes, in
both monovalent and multivalent configurations. With such an aim, the authors have
developed an immobilization strategy based on the mixing of two types of linkers,
one possessing the ability to covalently bind the biomolecules and the other being
unable to do it. Such an experimental setup, combined with an
ad hoc
analysis of
the unbinding properties, even by varying the relative concentrations of these two
linkers, has provided some general criteria to clearly distinguish between single and
multiple events. In this respect, the authors demonstrated that when multiple events
occur in parallel, the effective load applied to the system is reduced, and this should
be taken into account in the analysis of DFS data. Such an approach led them to find
out that the dissociation rate of the mucin 1-antibody complex drastically decreases
when multivalent unbinding processes occur, thus supporting the importance for the
biomolecules to preserve their multivalent capability for a more efficient binding. We
would like to remark that the developed immobilization strategy and the related data
analysis could be of rewarding help in the study of any ligand-receptor pairs.
Wojcikiewicz et al. have investigated the interaction of the leucocyte function-
associated antigen-1 with two different intercellular adhesion proteins ICAM-1 and
ICAM-2 involved in cell transmigration across endothelium (Wojcikiewicz et al.,
2006). Antigen-1 is a transmembrane glycoprotein, while ICAM-2 and ICAM-2 are
composed by immunoglobulin domains, a transmembrane domain and short cyto-
plasmic domain. Under normal conditions, ICAM-2 is the dominant receptor for
mediating leukocyte trafficking, whereas ICAM-1 becomes largely responsible for
mediating the adhesion of leukocytes to the inflamed endothelium. In DFS experi-
ments, antigen-1 molecules were directly embedded on the surface of Jurkat cells,
which were, in turn, bound to the AFM tip, whereas the antibodies were immobi-
lized on the substrate by a standard protocol (see Figure 6.6). Such an immobiliza-
tion strategy has the advantage of approaching the single-molecule regime while the
real physiological conditions are maintained. Both the complexes involving ICAM-1
or ICAM-2 show two distinct linear trends in the plot of the unbinding force as a
·
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