Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 38

screen in a Phosphorimager and analyze data using
ImageQuant software.
Compare the signal intensity obtained for a given dilution of
the viral stock to the plasmid DNA standard curve to cal-
culate the genome-containing particle titer in gcp/ml.

23

×

Y
N

× ×
×× ×

6 022 10
110

.

gcptiter = 1 000 ,

9

650

where Y is the amount of rAAV DNA in ng and N is the length
of the rAAV DNA template in base pair.
(b) ELISA
An enzyme-linked immunosorbent assay (ELISA) can be used
to determine the number of total particles (full and empty)
in a vector preparation. This assay uses a monoclonal anti-
body that specifically recognizes a conformational epitope
on assembled AAV capsids. The number of empty particles
can be subsequently determined by subtracting the num-
ber of genome-containing particles (obtained by dot blot
or q-PCR) from the number of total particles (obtained by
ELISA). The ratio of empty to full particles can therefore
be calculated for a given vector preparation.
Capsids titers for AAV1/6, AAV2/3, and AAV5 can be quanti-
fied using commercially available ELISA kits [ 63 , 64 ]. To
date, no kits are available for the other serotypes; however,
monoclonal antibodies recognizing assembled capsids for
AAV4, AAV8, and AAV9 have been generated to develop
serotype-specific ELISAs [ 24 , 64 ].
The AAV2 capsid titer is assessed using the AAV2 titration ELISA
kit, as briefly described below.
Prepare twofold serial dilutions of the AAV positive control in
450 μl ready to use buffer (1:1 to 1:64).
For viral samples, take 2.5 μl of virus in 247.5 μl ready to use buf-
fer and perform tenfold serial dilutions. For accurate assess-
ment of viral capsid titer, one of the dilutions of the viral
sample should be within the linear range of the ELISA (5 × 10 7
to 1 × 10 9 particles/ml). Perform each sample and control in
duplicate.
Add 100 μl of viral sample and standard serial dilutions to the
wells. Include a negative control (ready to use sample buffer).
Seal with adhesion foil and incubate for 1 h at 37 °C.
Add 200 μl of wash buffer per well. Incubate for approximately
5 s. Repeat washing step two more times.
Add 100 μl of biotin conjugate per well. Seal with adhesion foil
and incubate for 1 h at 37 °C. Repeat washing step.