Biomedical Engineering Reference
In-Depth Information
is inconsistent with what was expected and was attributed to the asymmetric struc-
ture of the IgG (Fc and F(ab
)
2
regions). If a highly idealized packing of antibody is
considered, the orientation of the adsorbed antibody can be estimated. In cases where
antibodies are oriented “end on” (Fc region closer to the SAM) or “head on” (F(ab
)
2
region closer to the SAM), the surface coverage is expected to range between 2.6 mg
m
2
and 5.5 mg m
2
. On the other hand, when antibodies are adsorbed “side on” (Fc
region and one Fab
region bound to the SAM), a surface coverage of approximately
2.0 mg m
2
is estimated. Based on these values, IgG bound to the COOH-terminated
SAMs is probably oriented head or end on. To further investigate the orientation of
adsorbed antibodies, the ratio of hCG to immobilized anti-hCG IgG was investigated
at various pHs. In these experiments, a higher ratio (0.48) was observed for IgG
adsorbed to the positively charged NH
2
surface near the IEP of IgG. This suggests
that there were more F(ab
)
2
regions exposed and available for analyte binding. In
summary, it was believed that IgG adsorbed to COOH-terminated SAMs has a head-
on orientation, and on the NH
2
-terminated SAMs the orientation is believed to vary
between end on and side on. Therefore, for immunosensing systems where capture
antibody adsorption to a SAM is the chosen method of immobilization, a positively
charged NH
2
-terminated SAM is the preferred surface. Additionally, adsorption should
be carried out under low ionic strength conditions and at a pH similar to the IEP of the
antibody.
Another way to control the orientation of a capture antibody on the surface of a
SAM is to employ Protein A or Protein G, covalently bound to the SAM. As described
in section 5.4.2, these proteins bind antibodies via their Fc regions and leave the
antigen binding sites available for binding analyte. Recently, our laboratory has
demonstrated the covalent coupling of Protein A to a thioctic acid SAM deposited on
a gold disk electrode [42]. In this work, the thioctic acid SAM was activated to its
o
-acylurea intermediate with EDC. A solution of Protein A (containing 30
g) was
then applied to the electrode surface. The electrode was incubated overnight at 4ºC to
facilitate the binding of Protein A via its lysine residues. The sensor surface was
then ready for incubation with capture antibody and subsequent processing of an
immunoassay.
µ
5.4.5 Antibody fragments
An alternative to immobilizing whole capture antibody molecules in immunoassay
systems is the immobilization of antibody fragments (Fab
). Fragmentation of an
antibody is usually achieved enzymatically with proteolytic enzymes such as chy-
motrypsin, trypsin, and papain [43]. Following enzymatic digestion, the disulfi de
linkages holding the two chains of the resulting F(ab
)
2
fragment together are typically
reduced with reagents such as dithiothreitol or 2-mercaptoethalamine. This results
in two Fab
fragments, each with a terminal thiol group. The fragments thus have
a high affi nity for a gold surface, on which they can therefore self-assemble without
the need of any additional reagent. The resulting layers have an ordered arrangement
with the antibody-binding regions being oriented in such a way that they are more
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