In the name of God

1. In the name of God

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2011

3. Ultracentrifugation & Ultrafiltration By: M. Shenagari A. Abdoli Summer School Influenza Unit, Pasteur Institute of Iran summer 2011

4. History and predecessors A 19th-century hand cranked laboratory centrifuge.English military engineer Benjamin Robins (1707–1751) invented a whirling arm apparatus to determine drag. In 1864, Antonin Prandtl invented the first dairy centrifuge in order to separate cream from milk. In 1879, Gustaf de Laval demonstrated the first continuous centrifugal separator, making its commercial application feasible. Influenza Unit, Pasteur Institute of Iran summer 2011

5. Influenza Unit, Pasteur Institute of Iran summer 2011

6. Protocols for centrifugation typically specify the amount of acceleration to be applied to the sample, rather than specifying a rotational speed such as revolutions per minute. This distinction is important because two rotors with different diameters running at the same rotational speed will subject samples to different accelerations. During circular motion the acceleration is the product of the radius and the square of the angular velocity ω, and the acceleration relative to "g" is traditionally named "relative centrifugal force" (RCF). Influenza Unit, Pasteur Institute of Iran summer 2011

7. The ability to culture cells in vitro has enabled enormous advances to be made in biology, particularly in virology

9. Components of basal media and their function

10. Main components of serum and their function

11. Why culture viruses? • The first is for the diagnostic identification of agents associated with disease. • The second is to enable some subsequent experimental manipulation of the virus to be performed, for example, to examine mechanisms of replication or to determine the effectiveness of potential antiviral strategies in vitro • The others: for example Vaccine development

12. Commonly used cell cultures

13. Uncultivable viruses HCV HBV HDV HPV,….

14. Concentration and purification ofviruses • Concentration: • Purification:

15. The need for virus concentration and purification • Virus infectivity or its neutralization by antibodies can usually be measured on non-concentrated preparations • 1 X 10 to powr 6 plaque-forming units of poliovirus may have a total mass of about 1 ng, of which 250 pg will be genomic RNA. While this is sufficient for genome amplification where the sequence is known

16. The need for virus concentration and purification more concentrated and purified preparations will be required for investigations such as: • physical analysis • genetic analysis where the genome sequence is not known • use as an antigen or immunogen

17. The need for virus concentration and purification • Concentrated virus preparations will contain the virus at a higher concentration than in the starting material but still include substantial amounts of impurities such as components from host cells. • These crude preparations can be used successfully in a variety of applications, including immunoprecipitation with specific antibodies, some routine antibody assays such as ELISA for known viruses, or as immunogens to produce neutralising antibodies. • Possible adverse effects of non-viral impurities can include false-positive reactions with antibodies, the induction of antibodies to non-viral antigens, or the detection of non-viral nucleic acids, and some purification procedure is usual following concentration.

18. The need for virus concentration and purification • Usually virus preparations would be concentrated before purification, but, where the virus occurs in high titre, it may be purified directly from the starting material

19. Example of sources and titres of material for virus purification

20. Properties of certain types of virus Influenza Unit, Pasteur Institute of Iran summer 2010

21. The quantities of starting material required will depend on the virus and the purpose for which it is intended, but it is advisable to select a virus strain and host cell which produce a high yield wherever possible. • The properties of the specific virus of interest may be a major factor in the details of the protocol used for concentration and purification

22. Purification and concentration points • Lipid-containing viruses, such as influenza, mumps, herpes, or hepatitis C virus, will be destroyed by organic solvents or detergents, • while non-lipid containing viruses, such as polio or SV40, may even require solvent or detergent treatment to remove contaminating cellular material in the course of purification.

23. Purification and concentration points • hepatitis C virus in human plasma has been reported to float in solution because of its association with lipid. • PH resistant or Labile • The size of virus particle • The density and sedimentation coefficient

24. Contaminants • lipids, • proteins, • and nucleic acids from host cells • as well as components of the matrix such as tissue culture medium, or plasma in which the virus is suspended

25. Virus concentration routine methods • Ultracentrifugation Equilibrium density gradients Velocity gradients Step gradients 2. Precipitation ammonium sulfate, PEG and sodium chloride 3. Ultrafiltration 4. column chromatography

26. Equilibrium density gradients • virus migrates until it reaches the position at which the density of the solution is the same as the density of the virus. • Lipid-containing viruses, such as retroviruses, may be purified on equilibrium sucrose density gradients, while polioviruses, being of higher density, require caesium chloride or sulfate to reach the correct density.

27. Velocity gradients • the virus moves at a rate determined by its sedimentation coefficient and the density of the gradient matrix

28. ultracentrifuge History • Theodor Svedberg invented the analytical ultracentrifuge in 1925,[1] and won the Nobel Prize in Chemistry in 1926 for his research on colloids and proteins using the ultracentrifuge. • The vacuum ultracentrifuge was invented by Edward Greydon Pickels. It was his contribution of the vacuum which allowed a reduction in friction generated at high speeds. Vacuum systems also enabled the maintenance of constant temperature. • In 1946, Pickels cofounded Spinco (Specialized Instruments Corp.) and marketed an ultracentrifuge based on his design. Pickels, however, considered his design to be complicated and developed a more “foolproof” version. But even with the enhanced design, sales of the technology remained low, and Spinco almost went bankrupt. The company survived and was the first to commercially manufacture ultracentrifuges, in 1947. In 1949, Spinco introduced the Model L, the first preparative ultracentrifuge to reach a maximum speed of 40,000 rpm. In 1954, Beckman Instruments (now Beckman Coulter) purchased the company, forming the basis of its Spinco centrifuge division. Influenza Unit, Pasteur Institute of Iran summer 2011

29. Purification by ultracentrifugation • The solute in the gradient is usually sucrose, caesium chloride, caesium sulfate, potassium sodium tartrate. • These reagents are chosen for their high solubility, and the high density of the resulting solutions.

30. Purification by ultracentrifugation

32. Step gradients • Step gradients involve layering a lower density solution onto a cushion of high density material.

33. A discontinuous sucrose density gradient is prepared by layeringsuccessive decreasing sucrose densities solutions upon one another. Influenza Unit, Pasteur Institute of Iran summer 2011

34. Influenza Unit, Pasteur Institute of Iran summer 2011

35. Density gradient maker for centrifuge tubes Influenza Unit, Pasteur Institute of Iran summer 2011

38. fit the original definition of “….principle of centrifugal force to separate materials of different densities,” specifically • HIGH SPEED centrifugesaka SUPERSPEED centrifuges and • ULTRA centrifuges up to around 22,000 rpm ultra = higher. Modern ultras have max speeds 80,000 – 150,000 rpm

39. High speed centrifuge • Beckman Avanti J series • 26,000 rpm (revolutions per minute) • 82,000 g (gravities) • Weight 600 lb

40. Ultracentrifuge • Beckman OptimaLXP • 100,000 rpm • 802,400 g • Weight 1025 lb

41. Mechanical Failure • Is caused by age and by improper use or inadequate care of centrifuge or rotor. Especially the rotor.

42. Rotors • Fixed angle rotor A high speed or ultra centrifuge rotor is a 10 - 30 lb piece of metal (aluminum and titanium are common), carefully designed and fashioned to turn smoothly and withstand the incredible forces concomitant with spin speeds of 15,000 - 150,000 rpm. • Swinging bucket rotor

43. Care and Attention • Safe high-speed spin requires nearly perfectly balanced load. • Age, use, and misuse contribute to rotor flaws. • A rotor which comes apart at high speed can be deadly.

44. Vivapure® Virus Purification and Concentration Kits

47. Ultrafiltration • Ultrafiltration is used to concentrate and also to purify virus from suspension, by removal of small molecular detritus which can pass through the filter, for example, serum proteins. • Filters are defined by their pore size, offering appropriate filter systems for different viruses

48. Ultrafiltration Membranes Ultrafiltration (UF) is the process of separating extremely small particles and dissolved molecules from fluids. The primary basis for separation is molecular size – particles ranging from 1,000 to 1,000,000 molecular weight are retained by ultrafiltration membranes Influenza Unit, Pasteur Institute of Iran summer 2011

49. Influenza Unit, Pasteur Institute of Iran summer 2011

50. Influenza Unit, Pasteur Institute of Iran summer 2011