Chemical, Inc., Natick, MA, USA) for 1 h at room
temperature followed by trypsin treatment 0.05% for
1 h in the presence of 1% Tween-80. The resulting lysate
was ®ltered through a 0.8 mm Millex HV ®lter unit before
chromatography. Routinely 10±15 roller bottles were
harvested equivalent to 1±5 e 9 cells.
BioRad ceramic hydroxyapatite
(80 mm)
Cell lysates from 293 cells infected with adeno-associated
virus (in the presence of Ad5ts149) were chromato-
graphed on a hydroxyapatite resin (40 ml) which was pre-
equilibrated with 10 mM sodium phosphate pH 6.4,
containing 10 mM NaCl, 0.1% Tween-80, 10% glycerol
and 2 mM MgCl
2
. rAAVbgal was applied to the resin in the
same buffer. Bound proteins were eluted from the resin
using a linear salt gradient (120 ml) of 10±400 mM
sodium phosphate at pH 6.4. Fractions collected from the
resin were assayed for rAAVbgal proteins by immuno-
blotting using an antibody against the three capsid
proteins of rAAVbgal, VP1, VP2 and VP3 (Catalog 03-
65158, American Research Products, Belmont, MA, USA).
Fractions eluted from the resin were also assayed for
adenoviral-contaminating proteins by immunoblotting
using an anti-adenoviral antibody.
DEAE chromatography
A DEAE Macroprep resin (BioRad) (5 ml) was equili-
brated with 10 mM sodium phosphate buffer containing
50 mM NaCl, 10% glycerol, pH 7.5. rAAVbgal-containing
fractions eluted from the hydroxyapatite column were
pooled and dialyzed into the same buffer used for
equilibration of the DEAE resin. A linear gradient
(50 mM±1 M NaCl in 10 mM sodium phosphate, pH 7.5,
10% glycerol, 0.05% Tween-80) was applied to the resin
at a ¯ow rate of 5 ml/min, the volume of the gradient was
50 ml. Bound proteins were eluted from the resin and
collected in 2.5-ml fractions. Each fraction was assayed
for rAAVbgal proteins and contaminating adenoviral
proteins (Coomassie blue staining and immunoblotting).
In addition all fractions were assayed for rAAVbgal and
Ad5ts149 infectivity by titer analysis. Fractions which
were positive for both rAAVbgal protein and infectivity
were pooled and chromatographed further using a
Cellu®ne sulphate resin.
Cellu®ne
1
sulphate resin (Amicon)
Cellu®ne sulphate resin (3 ml column volume) was
equilibrated with phosphate buffer saline (PBS) contain-
ing 10% glycerol. Fractions eluted from the DEAE resin
containing both rAAVbgal proteins and DNA were pooled
and applied to the resin at a ¯ow rate of 4 ml/min. The
resin was washed with 250 mM NaCl and a linear salt
gradient (0.25±1 M NaCl in PBS/10% glycerol) was
applied. The volume of the gradient was 10 ml. The
eluted fraction and the ¯ow through were analyzed for
both rAAVbgal and Ad5ts149 proteins (immunoblotting)
and infectivity (titer analysis). The ®nal fraction was also
assayed for rAAVbgal DNA by slot blot analysis.
SDS-PAGE analysis
One-dimensional SDS-PAGE was performed using
10±20% gradient (Daiichi) gels. Samples were boiled
for 5 min in SDS-PAGE reducing buffer 125 mM Tris-HCl,
pH 6.8, 20% glycerol, 4% (wt/vol) SDS, 0.05% bromo-
phenol blue, 0.5% b-mercaptoethanol before loading on
gel. Proteins in the gel were detected using Coomassie
blue or silver stain. For immunoblotting, PVDF mem-
branes (Novex) were pre-wetted with methanol and
soaked in 10 mM CAPS, pH 11.0 containing 10%
methanol. Gels were equilibrated in this transfer buffer
for 10 min and then blotted at 30 V for 1 h in a Novex Blot
Module. After transfer membranes were blocked with 1%
dried milk in TBS (20 mM Tris-HCl, pH 7.5 containing
150 mM NaCl) for 1 h. After blocking, the membranes
were probed with anti-VP1, VP2, VP3 antibody or anti-
adenoviral antibody in 20 mM Tris-HCl, 150 mM NaCl,
pH 7.5 and 0.05% Tween 20 (TBST) containing 0.1% BSA
for 1 h. The membranes were incubated with horseradish
peroxidase labeled anti-mouse or anti-goat IgG for 45 min
and the immunoreactive bands visualized by chemilumi-
nescence using the BM Chemiluminescent Western
Blotting Detection System (Boehringer Mannheim).
Measurement of infectious particles
The rAAVbgal titer was determined by an end-point
dilution assay on 293 cells. In brief, 293 cells were plated
into a 96-well micro-titer plate, 100 ml of 5 e 5 cells/ml
for each well. In a separate plate a 200-ml aliquot of virus
sample diluted 1 : 100 was added to the ®rst column and
was serially diluted two-fold across the plate. A 100 ml-
aliquot of each well was transferred to its identical
position in the 293 seeded plate and allowed to incubate
at 37uC in a humidi®ed air 5% CO
2
incubator for 2 days.
The media was removed by aspiration and the cells were
stained with X-gal (5 bromo-4-chloro-3-indol-b-D-
galactopyranoside) for 20±24 h. Titers were calculated
with a computer program based on Karber's method [33].
The titer of contaminating Ad5ts149 was determined
using a similar end-point dilution assay except staining
was for hexon using ¯uorescence isothiocyanate (FITC)-
conjugated anti-hexon antibody [34].
Slot blot analysis of column fractions
for detection of rAAV DNA
Column fractions were assayed for rAAVbgal DNA by slot
blot analysis as described by Vincent et al. [20].
446 C. R. O'Riordan et al.
Copyright # 2000 John Wiley & Sons, Ltd. J Gene Med 2000; 2: 444±454.
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