Biomedical Engineering Reference
In-Depth Information
Fig. 3
Proximity ligation assay to study virus-host interactions. (
a
) A schematic
diagram of the proximity ligation assay for the detection of virus localization to
subcellular structures. (
b
) HeLa cells were infected with HPV16-GFP at MOI of
100 (
right panel
) or remained uninfected (
left panel
). Sixteen hours later, cells
were stained with the L1 polyclonal antiserum and the GM130 Golgi-specifi c
antibody and processed for PLA. A single confocal slice is shown in each panel.
PLA signal is
green
. Nuclei are stained
blue
with DAPI. Reproduced from ref.
5
with permission from the Proceedings of the National Academy of Sciences, USA
In fact, commercially available kits based on PLA technology
nominally allow the detection of proteins separated by 40 nm
(approximately the diameter of a small virus capsid) [
14
]. PLA in
effect converts antibody-mediated recognition of proximal epit-
opes into an amplifiable DNA sequence. Rather than being
precipitated, the assembly of proteins, bound antibodies, nucleic
acid aptamers, and amplifi cation products is labeled in situ with a
large number of fl uorescent DNA probes, resulting in high sen-
sitivity (Fig.
3a
). Typically, to visualize incoming virus during
infection studies, one antibody is directed against a virion protein
and the other to a cellular protein localized in a particular com-
partment (or to a required hit on a siRNA screen). A successful
proximity ligation assay for the presence of a virus in a particular
