Biomedical Engineering Reference
In-Depth Information
The data of this study demonstrate that ANA-BA is a test system with great
potential for routine diagnostics of systemic rheumatic diseases. The analytic
performance and the test handling are comparable with standard ELISA tests that
were used as references for each analytic parameter. The test precision and sta-
bility of ANA-BA were also found to be in the range of diagnostic semiquanti-
tative ELISA tests.
4.3 Immobilization and Detection of Biotinylated Antigens
from Crude Bacterial Lysates
The development of multi-parameter microbead-based assays requires the
immobilization not only of DNA capture probes; proteins or peptides are equally
important to serve as capture probes for the detection of autoantibodies in patient
sera or for epitope mapping. Different biomolecule immobilization strategies,
including passive adsorption and covalent attachment, have been described pre-
viously [
47
,
48
]. Binding of biotinylated capture probes to streptavidin-coated
microbeads is also an elegant approach. The high affinity and specificity of the
biotin-streptavidin interaction is widely used for biomolecule labeling or for
purification purposes [
49
]. A major drawback of this approach is the tedious, time-
consuming and expensive production of these essential biotinylated antigens.
Instead of protein/peptides synthesis and chemical biotinylation, we aimed for a
fast, easy and inexpensive alternative approach using a technology first described
by Beckett et al. [
50
] and patented as AviTag
TM
technology (Avidity) (Fig.
13
a).
In this, peptides are expressed as fusion peptides with an AviTag
TM
(Fig.
13
b).
This tag is a 15-amino-acid peptide sequence with a lysine residue, which is
biotinylated by a biotin ligase. The biotin ligase is overexpressed in Escherichia
coli cells (Fig.
13
c). Biotinylated peptides can be directly immobilized on
streptavidin-coated microbeads merely by disrupting cells and incubation with
the lysate. Unbound material is subsequently removed by washing. In a proof-
of-principle approach, we used the well-characterized glutathione S-transferase
(GST) as model protein.
In contrast to the notion that the expression of biotinylated proteins using
the AviTag
TM
is not suitable [
51
] because the overexpression of a biotin ligase in
E. coli causes cell damage and in efficient biotinylation, we observed a func-
tionally sufficient biotinylation efficiency of different peptides (data not shown).
Biotinylation takes place very specifically since only the AviTag
TM
is biotinylated
but not the fusion protein itself. This should minimize the risk of severely affecting
binding properties by peptide modifications. Since there is only one biotinylation
site per tagged peptide, it is well orientated in regard to the microbead surface and
it is easily accessible for potential binding partners compared to randomly bio-
tinylated or covalently immobilized molecules, which are attached to microbeads
via many different binding sites. The ease of this method, in addition to time and
cost savings, makes it very attractive for use with VideoScan.
