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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. 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

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  1. Virus purification and analysis using OptiPrep™ - competitive media • Sucrose • Glycerol • Caesium chloride

  2. 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

  3. 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

  4. 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

  5. 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

  6. Virus banding density

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 1 2 3 3 Formats for separation of particles accordingto their density

  13. 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

  14. Comparison of rAAV in CsCl and OptiPrep™ Data from Hermans, WTJMC et al (1999) Human Gene Therapy, 10, 1885-1891

  15. 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

  16. Use of self-generated iodixanol gradients in vertical rotors for virus purification and analysis

  17. 1 2 3 50,000g 1h 350,000g 4 5 1-3h Virus purification flow-chart – without virus pelleting

  18. 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)

  19. 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

  20. 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:

  21. 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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