Biomedical Engineering Reference
In-Depth Information
100
H
½
%
¼
1
H
1
H
0
ð
3
Þ
3.4 Determination of Functional Capture Probe Density
To perform assays with a good sensitivity and specificity it is important to
determine the density of immobilized capture probes that is useful for VideoScan
assays. It is well known that the surface capture probe density is negatively cor-
related with the hybridization efficiency (''H'') of oligonucleotides probes. A high
density may lead to steric hindrance, the so called ''hook effect'' [
28
].
To determine the range of functionally immobilized capture probe densities, a
standard hybridization FRET assay was applied (Fig.
7
). Again, three microbead
populations were loaded with low, medium and high quantities of Atto 647N-
labeled capture probes MLC-2v-cap or aCS-cap. The complementary target
sequence for MLC-2v-cap or aCS-cap, respectively, was added at a final con-
centration of 150 nM. The samples were set to hybridize initially at 95 C for 2.5
min and finally at 60 C for 60 min. Under these conditions we found again that
the minimum number of microbeads is 20 per population. Although showing an
increased signal-to-noise ratio, microbeads with a medium or high capture probe
density reported higher signal variability at low microbead numbers (\80), which
was not the case for microbeads of lower capture probe density. Increasing the
number of beads measured could easily compensate for this effect (Fig.
9
c, d). We
therefore conclude that VideoScan assays designed with microbeads of variable
capture probe densities do not suffer from the adverse hook effect [
28
]. This
broadens the range of suitable microbead populations and enables a multi-purpose
assay design.
4 VideoScan Technology Applications
4.1 AKLIDES
Application for the Automated Detection
of Autoantibodies by Cell-Based Assays
Based on VideoScan technology development, an automated indirect immuno-
fluorescence (IIF) microscopy system for the assessment of autoantibodies (AAB)
employing cell- and microbead-based assays was recently commercialized under
the brand name AKLIDES
(Medipan GmbH, Dahlewitz/Berlin, Germany) [
29
].
Autoantibodies play a pivotal role in the serological diagnosis of systemic rheu-
matic and autoimmune liver diseases [
30
-
32
]. Autoantibody assessment is used to
monitor disease activity, sub-classify, and predict autoimmunity [
33
,
34
].
Modern laboratory standards require high reproducibility and standardization of
IIF interpretation which is difficult to achieve due to subjective visual reading by
