Biomedical Engineering Reference
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low anti-
a
-subunit titers [19]. Taken together, these data
suggest that the
a
-AChR portion of SHG2210 adopts a
native or near native conformation in solution and is capable
of binding to a variety of
a
-subunit specific anti-AChR
antibodies. The fusion protein therefore performs as engi-
neered with respect to antibody binding.
HeLa cells was transferrin receptor specific, as demonstrated
by negligible uptake in the presence of saturating levels of
human transferrin as inhibitor [19]. ELISA results were
further verified by molecular imaging data. Imaging experi-
ments were performed in the human muscle cell line DB40,
a transfected subclone of the TE671 muscle cell line fre-
quently used for in vitro studies of human MG disease [22],
in order to more closely approximate the cells of interest in
actual MG disease. By directly labeling both fusion protein
and anti-AChR antibody with fluorescent dyes having dis-
tinct emission wavelengths (Alexa fluor 564 and 488,
respectively), the fusion protein:antibody complexes bound
to transferrin receptor within cells was visualized using
three-color laser scanning confocal microscopy (Figure
12.3). Therefore, using a combination of biochemistry
and fluorescent imaging, the fusion protein:antibody com-
plex was shown to be taken into cells via TFR.
12.3.3 SHG2210: Anti-AchR Antibody Complex
Binding and Internalization Studies in Cells
SHG2210:antibody complex binding and uptake by HeLa
cells was first assayed by ELISA. The ELISA used antihu-
man transferrin as a capture antibody and peroxidise-
conjugated anti-rat IgG as detection antibody. SHG2210:
antibody complexes were preformed before adding to cells
using mAb35 as model anti-AChR antibody. As with free
fusion protein, fusion protein:antibody complex uptake by
FIGURE 12.3
Molecular imaging of internalized SHG2210:mAb 35 complex in human muscle
cells by confocal microscopy SHG2210 fusion protein and mAb 35 were directly labeled with Alexa
fluor 546 and 488 dyes, respectively. Fusion protein and antibody were preincubated for 15min to
form complexes and then added to the human muscle cell line DB40 for 2 h on ice. Cells were then
washed and switched to 37
C to initiate uptake of the complex. Uptake of complex was allowed to
occur for 15 min, at which point cells were fixed and processed for immunofluorescence using
antitransferrin receptor monoclonal antibody. Clockwise from top: anti-TF receptor (blue); mAb 35
(green); SHG2210 (red); merged image.
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