virus purification and analysis using optiprep™ - competitive media sucrose glycerol caesium...
TRANSCRIPT
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Virus purification and analysis using OptiPrep™ - competitive
media
• Sucrose
• Glycerol
• Caesium chloride
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SOLUTION HANDLING
• Density solutions prepared by diluting OptiPrep™ 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
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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
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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
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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
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Virus banding density
Virus CsCl Sucrose Iodixanol
Epstein-Barr 1.26 1.14
Semliki Forest 1.26 1.18 1.18
Poliovirus 1.34 1.26 1.22
Measles 1.21 1.18 1.16
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Kivela et al (1999)Virol. 262, 364-374
0 10 20 30 40 50 60
% iodixanol, sucrose, glycerol
1011
1012
1.1 1.3 1.5 1.7 CsCl g/ml
Iodixanol
Glycerol
CsClSucrose
Infe
ctiv
ity
Effect of medium on PM2 infectivity
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• Sucrose gradients cause loss of virus surface glycoproteins; no loss with iodixanol gradients
• Infectivity recoveries are 10-100x greater in iodixanol than in sucrose
SUCROSE PROBLEMS (II):for enveloped viruses,
particularly retroviruses
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15% iodixanolin 1 M NaCl
25% iodixanol
40% iodixanol
54% iodixanol
Virus fluid
proteins
adenovirus
rAAV
350,000g 1 h
rAAV in discontinuous gradient (V04)Zolotukhin S. et al (1999) Gene Therapy, 6, 973-985
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rAAV in continuous gradient (V04)Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy,
10, 1885-1891
Opti-Prep
Virusfluid
B
Gradient Master
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Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of
timeDensity (g/ml)
Fraction Number1 3 5 7 9
1.07
1.09
1.11
1.132 min2.5 min3.0 min
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3 Formats for separation of particles accordingto their density
1
2
3
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rAAV in continuous gradient (V04)Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy,
10, 1885-1891
348000g3 h
rAAV
mem
bra
nes
OptiPrep
Virusfluid
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Comparison of rAAV in CsCl and OptiPrep™
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 GeneTherapy, 10, 1885-1891
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Purification of HIV-1 (V06)Dettenhoffer, M & Yu, X-F (1999) J. Virol., 73 1460-1467
250,000g for 1.5 h
6-18% iodixanolMicrovesiclesVif gene
Soluble proteins
RT (cpmx10-5/ml)
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Use of self-generated iodixanol Use of self-generated iodixanol gradients in vertical rotors for gradients in vertical rotors for virus purification and analysisvirus purification and analysis
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1 3
45
250,000g
1h
350,000g
1-3h
Virus purification flow-chart –without virus pelleting
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Purification of Herpes virus in self-generated 25% iodixanol gradient (V05)
Fraction Number
Log
infe
ctiv
ity
Den
sity
(g/
ml)
1 3 5 7 9 11 13 150
1
2
3
4
5
6
1
1.05
1.1
1.15
1.2
1.25
1.3Infectivity Density
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Fraction number
OD
(PE
RT
) x
10-2
Den
sity
(g
/ml)
1 3 5 7 9 11 13 15 17 190
5
10
15
20
25
30
1
1.05
1.1
1.15
1.2
1.25MS1533MS1874 Density
Purification of retrovirus: self-generated gradient (V09)Moller-Larsen A & Christensen, T. (1998) J. Virol Meth., 73, 151-161
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OptiPrep™Application 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
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Publications database on virusesPublications 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