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 • 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 1.14

Semliki Forest 1.26 1.18 1.18

Poliovirus 1.34 1.26 1.22

Measles 1.21 1.18 1.16

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

• 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

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

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

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

3 Formats for separation of particles accordingto their density

1

2

3

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

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

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)

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

1 3

45

250,000g

1h

350,000g

1-3h

Virus purification flow-chart –without virus pelleting

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

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

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

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