Biomedical Engineering Reference
In-Depth Information
the surface of a pre-treated SPCE (Fig. 5.5b). This assay format yielded a detection
limit of 40 ng mL
1
, indicating that the biotin-streptavidin interaction used to immobi-
lize capture antibody provides a suitable support for electrochemical immunosensing.
Further, it is likely that suffi cient capture antibody was immobilized on the surface of
the SPCE with appropriate orientation for binding analyte.
5.4.2 Antibody-binding proteins
Another commonly used affi nity-based immobilization technique for capture anti-
bodies in immunoassay systems involves a bacterial antibody-binding protein. The
two most common of which are Protein A and Protein G. These proteins bind specifi -
cally to antibodies through their non-antigenic (Fc) regions, which allow the antigen
binding sites of the immobilized antibody to be oriented away from the solid phase
and be available to bind the target analyte. As these proteins interact directly with the
Fc region of antibodies, there is no need for antibody biotinylation. Protein A has a
molecular weight of approximately 42 kDa and was originally isolated from the cell
wall of
Staphylococcus aureus
[28]. It is known to contain fi ve Fc binding domains
located towards its
ß
NH
2
terminal. However, the binding capacity of Protein A is lim-
ited to three human IgG subclasses (IgG 1, 2 and 4) [29]. Also, Protein A will not
bind to goat and rat IgG, and only weakly to mouse IgG [30]. The second bacterial
antibody binding protein, Protein G, is a cell surface protein of group C and G
strep-
tococci
with three Fc binding domains located near its C-terminal, and has specifi city
for subclasses of antibodies from many species [29]. In 2004, Zacco
et al.
reported
a rigid material for use as a scaffold in electrochemical immunosensing that is based
upon a Protein A bulk-modifi ed graphite-epoxy biocomposite (Protein A-GEB) [31].
This biocomposite not only provides a means to securely immobilize the capture anti-
body, but also acts as the transducer for the electrochemical signal. The biocomposite
layer was formed by mixing graphite powder with epoxy resin in a 1:4 (w/w) ratio,
followed by adding Protein A to a fi nal concentration of 2% (w/w). The resulting paste
was then placed in a cylindrical sleeve body with electrical contact and the Protein
A-GEB was cured for a week. The suitability of this layer as a scaffold for electro-
chemical immunosensing was investigated with a model competitive immunoassay. A
schematic representation of the assay system is represented in Fig. 5.6. First, rabbit
antibody (RIgG) was introduced to the layer and allowed to interact with Protein A via
its Fc regions. Biotinylated anti-RIgG was then introduced to bind to the immobilized
RIgG. Streptavidin-labeled HRP was introduced to bind to the bound anti-RIgG before
processing the immunoassay by introducing the substrate H
2
O
2
. The assay could dis-
tinguish between 2 pmol and 10 pmol of anti-RIgG. These workers have also shown
that the Protein A-GEB layer can be regenerated by polishing with abrasive and alu-
mina papers, which yields a smooth mirror fi nish containing freshly exposed Protein A
that may be reused in subsequent assays.
By applying a solution with the appropriate pH and ionic strength, the interac-
tion between Protein A or Protein G and the antibody can be reversed, enabling easy
renewal of sensing surfaces [32, 33]. This has been demonstrated by Yakovleva
et al.
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