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Virus purification and analysis using OptiPrep ™ - competitive media. Sucrose Glycerol Caesium chloride. SOLUTION HANDLING. Density solutions prepared by diluting OptiPrep ™ with culture medium or any balanced salt solution (Application Sheet V01) Sterile solution

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virus purification and analysis using optiprep competitive media
Virus purification and analysis using OptiPrep™ - competitive media
  • Sucrose
  • Glycerol
  • Caesium chloride
solution handling
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
cscl problems
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
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
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
slide7

CsCl g/ml

1.3

1.1

1.5

1.7

Iodixanol

1012

Infectivity

Glycerol

1011

CsCl

Sucrose

0

10

20

30

40

50

60

% iodixanol, sucrose, glycerol

Kivela et al (1999)Virol. 262, 364-374

Effect of medium on PM2 infectivity

sucrose problems ii for enveloped viruses particularly retroviruses
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
slide9

Virus fluid

15% iodixanol

in 1 M NaCl

proteins

25% iodixanol

adenovirus

40% iodixanol

rAAV

54% iodixanol

rAAV in discontinuous gradient (V04)

Zolotukhin S. et al (1999) Gene Therapy, 6, 973-985

350,000g 1 h

slide10

Virus

fluid

B

Opti-Prep

Gradient Master

rAAV in continuous gradient (V04)

Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy, 10, 1885-1891

gradient master profiles from 10 and 40 iodixanol at 80 and 20 rpm effect of time

Density (g/ml)

2 min

1.13

2.5 min

3.0 min

1.11

1.09

1.07

1

3

5

7

9

Fraction Number

Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time
raav in continuous gradient v04 hermans w t j m c et al 1999 human gene therapy 10 1885 1891

Virus

fluid

348000g

3 h

membranes

OptiPrep

rAAV

rAAV in continuous gradient (V04)Hermans, W.T.J.M.C. et al (1999) Human Gene Therapy, 10, 1885-1891
comparison of raav in cscl and optiprep
Comparison of rAAV in CsCl and OptiPrep™

Data from Hermans, WTJMC et al (1999) Human Gene

Therapy, 10, 1885-1891

slide15

Purification of HIV-1 (V06)

Dettenhoffer, M & Yu, X-F (1999) J. Virol., 73 1460-1467

RT (cpmx10-5/ml)

Soluble proteins

6-18% iodixanol

Microvesicles

250,000g for 1.5 h

Vif gene

slide17

1

2

3

50,000g

1h

350,000g

4

5

1-3h

Virus purification flow-chart –

without virus pelleting

purification of herpes virus in self generated 25 iodixanol gradient v05

Infectivity

1.3

6

Density

1.25

5

1.2

4

Density (g/ml)

Log infectivity

1.15

3

1.1

2

1.05

1

1

0

1

3

5

7

9

11

13

15

Fraction Number

Purification of Herpes virus in self-generated 25% iodixanol gradient (V05)
slide19

30

1.25

MS1533

MS1874

25

Density

1.2

20

1.15

OD(PERT) x 10-2

Density (g/ml)

15

1.1

10

1.05

5

0

1

1

3

5

7

9

11

13

15

17

19

Fraction number

Purification of retrovirus: self-generated gradient (V09)

Moller-Larsen A & Christensen, T. (1998) J. Virol Meth., 73, 151-161

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

OptiPrep™Application Sheets for:
publications database on viruses
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