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Methods for the purification of viruses and analysis of virus processing

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Virus purification and analysis
using OptiPrepв„ў - competitive
media
• Sucrose
• Glycerol
• Caesium chloride
SOLUTION HANDLING
• Density solutions prepared by diluting
OptiPrepв„ў with culture medium or any
balanced salt solution (Application Sheet
V01)
• Sterile solution
• CsCl/sucrose solutions require lengthy
preparation times and sterilization
• CsCl is expensive
CsCl PROBLEMS
• Big losses in viral infectivity; low recoveries;
high particle:infectivity ratios
• Solutions have very high osmolality: viruses
lose water; high virus density; high [CsCl]
required for gradients
• Highly toxic to cells: must be dialyzed prior
to re-infection of cells
• Solutions ionic and corrosive: samples must
be dialyzed before electrophoresis or HPLC
SUCROSE PROBLEMS (I)
• Solutions are hyperosmotic: virus density
in sucrose usually higher than in
iodixanol
• Solutions are viscous: slow sedimentation
of virus particles
• Highly toxic to cells: must be dialyzed
prior to re-infection of cells
OPTIPREP – NO PROBLEMS
• Solutions are isoosmotic: virus density is
low
• Solutions have low viscosity: rapid
sedimentation of virus particles
• Is non-toxic to cells
• Has little or no effect on virus infectivity
• Cells can be re-infected and electrophoresis
and HPLC performed without dialysis
Virus banding density
Virus
CsCl
Sucrose
Iodixanol
Epstein-Barr
1.26
Semliki Forest
1.26
1.18
1.18
Poliovirus
1.34
1.26
1.22
Measles
1.21
1.18
1.16
1.14
Effect of medium on PM2 infectivity
Infectivity
1.1
1.3
1.5
1.7 CsCl g/ml
Iodixanol
1012
Glycerol
1011
CsCl
Sucrose
0
10 20 30 40 50 60
% iodixanol, sucrose, glycerol
Kivela et al (1999)Virol. 262, 364-374
SUCROSE PROBLEMS (II):
for enveloped viruses,
particularly retroviruses
• Sucrose gradients cause loss of virus
surface glycoproteins; no loss with
iodixanol gradients
• Infectivity recoveries are 10-100x
greater in iodixanol than in sucrose
rAAV in discontinuous gradient (V04)
Zolotukhin S. et al (1999) Gene Therapy, 6, 973-985
Virus fluid
15% iodixanol
in 1 M NaCl
350,000g 1 h
25% iodixanol
proteins
40% iodixanol
54% iodixanol
adenovirus
rAAV
rAAV in continuous gradient (V04)
Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy,
10, 1885-1891
Virus
fluid
B
OptiPrep
Gradient Master
Gradient Master profiles from 10% and
40% iodixanol at 80В° and 20 rpm: effect of
time
Density (g/ml)
1.13
2 min
2.5 min
3.0 min
1.11
1.09
1.07
1
3
5
7
Fraction Number
9
3 Formats for separation of particles according
to their density
1
2
3
rAAV in continuous gradient (V04)
Virus
fluid
348000g
3h
membranes
Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy,
10, 1885-1891
OptiPrep
rAAV
Comparison of rAAV in CsCl and
OptiPrepв„ў
Pre-gradient
Gradient
% Recov’ry
VP/ICU
(NH4)2 SO4
CsCl
0.1-60
250-108
(NH4)2 SO4
OptiPrep
26-120
264-1333
Cellufine-SO4
OptiPrep
25-91
385-600
Data from Hermans, WTJMC et al (1999) Human Gene
Therapy, 10, 1885-1891
Purification of HIV-1 (V06)
Dettenhoffer, M & Yu, X-F (1999) J. Virol., 73 1460-1467
RT (cpmx10-5/ml)
6-18% iodixanol
250,000g for 1.5 h
Soluble proteins
Microvesicles
Vif gene
Use of self-generated iodixanol
gradients in vertical rotors for
virus purification and analysis
Virus purification flow-chart –
without virus pelleting
1
50,000g
2
1h
5
4
350,000g
1-3h
3
Purification of Herpes virus in selfgenerated 25% iodixanol gradient (V05)
Log infectivity
6
1.3
5
1.25
4
1.2
3
1.15
2
1.1
1
1.05
0
1
3
5
7
9
11
Fraction Number
13
15
1
Density (g/ml)
Infectivity
Density
Purification of retrovirus: self-generated gradient (V09)
Moller-Larsen A & Christensen, T. (1998) J. Virol Meth., 73, 151-161
MS1533
MS1874
Density
OD(PERT) x 10-2
25
1.25
1.2
20
1.15
15
1.1
10
5
1.05
0
1
1
3
5
7 9 11 13 15 17 19
Fraction number
Density (g/ml)
30
OptiPrepв„ўApplication Sheets for:
•
•
•
•
•
•
•
•
•
Adenovirus
Ebola virus
Foamy virus
Herpes simplex virus
HIV-1
Human papillomavirus
Lassa virus
Murine leukemia virus
Murine oncornavirus
•
•
•
•
•
•
•
•
•
Norwalk virus
Polyoma virus
rAAV
Rabies virus
Rous sarcoma virus
Retroviruses
Semliki Forest virus
SARS Corona virus
Vaccinia virus
Publications database on viruses
• OptiPrep (since 1994) over 600
• Nycodenz® (since 1984) approx 70
• Using either the Applications CD or the
website:
• www. axis-shield-density-gradient-media.com
• Follow the instructions to access the relevant
Index
• Click on the virus of interest
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