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In the name of God
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Summer School
Influenza Unit,
Pasteur Institute of Iran
summer 2011
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Summer School
Ultracentrifugation & Ultrafiltration
By: M. Shenagari
A. Abdoli
Influenza Unit, Pasteur Institute of Iran
summer 2011
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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
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Influenza Unit, Pasteur Institute of Iran
summer 2011
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• 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
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The ability to culture cells in vitro has enabled
enormous advances to be made in biology,
particularly in virology
Culture collection
Address
ECACC
European Collection of Cell Cultures
Centre for Applied Microbiology & Research, Salisbury, Wiltshire
SP4 0JG, UK
http://www.camr.org.uk/ecacc.htm/
ATCC
American Type Culture Collection
P.O. Box 1549, Manassas, Virginia 20108, USA
http://www.atcc.org/
DSMZ
Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH
Mascheroder Weg 1B, D-3300 Braunschweig, Germany
http://www.dsmz.de/
Riken
Riken Cell Bank
3-1-1 Koyadai, Tsukuba Science City, 305 Iboraki, Japan
http://www.rtc.riken.go.jp/
Components of basal media and their function
Component
Function
Balanced salt solution
Maintain physiological pH, maintain osmotic pressure, membrane
potential, cofactors for enzymes
Buffering systems
e.g. Bicarbonate/CO2, Hepes
Compensate for C02 and lactic acid production; HCO3 is also a growth
factor
Carbohydrates or glutamine
e.g. glucose, galactose
Energy source
Amino acids
Essential amino acids not synthesised by cells, non-essential amino
acids which may be lost by cells into medium
Vitamins
e.g. para-aminobenzoic acid, biotin, folic acid, B12, riboflavin etc.
Precursors for cofactors
Hormones and growth factors
e.g. insulin, hydrocortisone, nerve growth factor, epidermal growth
factor, fibroblast growth factor, etc.
Stimulate cell proliferation or differentiation
Proteins and peptides
e.g. fetuin, a-globulin, fibronectin, albumin, transferrin
Carry hormones, vitamins, lipids, etc.
Fatty acids and lipids
Membrane biosynthesis, etc
Accessory factors
e.g. trace elements, nucleotides
Enzyme co-factors, etc.
Main components of serum and their function
Component
Function
Growth Factors
Stimulate cell proliferation or differentiation.
Albumin
Carrier protein for small molecules
e.g. lipids, steroids, vitamins, metal ions
pH buffer; protects cells against mechanical
damage in agitated systems
Transferrin
Iron transport
Anti-proteases, e.g. a1 antitrypsin, a2
macroglobulin
Prevent proteolytic damage to cells.
Attachment factors, e.g. fibronectin, fetuin,
laminin
Allow binding of attachment dependent cells
to substrate.
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
Commonly used cell cultures
Type of culture
Viruses capable of replication
Primary/secondary cultures
Monkey kidney cells
Influenza viruses,
parainfluenza viruses,
enteroviruses,
mumps virus
Semi-continuous cell lines
Human embryonic fibroblasts
Herpes simplex viruses (HSV),
varicella zoster virus,
cytomegalovirus,
enteroviruses,
adenoviruses,
rhinoviruses
Continuous cell lines
Vero cells (derived from monkey kidney)
HEp-2 cells
HeLa cells
HSV,
mumps virus
Respiratory syncytial virus,
adenoviruses
Adenoviruses
Uncultivable viruses
HCV
HBV
HDV
HPV,….
Concentration and purification of
viruses
• Concentration:
• Purification:
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
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
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 falsepositive reactions with antibodies, the induction of antibodies to nonviral antigens, or the detection of non-viral nucleic acids, and some
purification procedure is usual following concentration.
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
Example of sources and titres of material for virus purification
Virus
Source
Titre
Rotavirus
Faecal material
10^12 particles ml-1
Parvovirus B19
Blood plasma (acute phase)
10^12 particles ml-1
Hepatitis C
Plasma (acute phase)
10^6 genome equivalents ml-1
Hepatitis C
Plasma (chronic phase)
10^4 genome equivalents ml-1
Influenza
Allantoic fluid from chicken eggs
10^9 infectious units ml-1
Mumps
Allantoic fluid from chicken eggs
10^6 infectious units ml-1
Polio
Tissue culture fluid
10^8 infectious units ml-1
SV40
Tissue culture fluid
10^8 infectious units ml-1
Mumps
Tissue culture fluid
10^4-10^6 infectious units ml-1
Rubella
Tissue culture fluid
10^4-10^6 infectious units ml-1
Properties of certain types of virus
Virus
envelope Density(g cm-3) Sedimentation coefficient (S)
Parvovirus
no
1.39-1.42
110-120
Poliovirus
no
1.34
160
SV40
no
1.2a-1.34b
240
Rubella
yes
1.18
280
Yellow fever
Yes
1.23
170-210
Hepatitis C
yes
1.09-1.11
>150
Influenza
yes
1.19
700-800
Mumps
yes
1.18-1.20
>1000
Murine leukaemia
virus
yes
1.16-1.18
700-800
Herpes virus
yes
1.20-1.29
>1000
Influenza Unit, Pasteur Institute of Iran
summer 2010
• 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
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.
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
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
Virus concentration routine methods
1. Ultracentrifugation
Equilibrium density gradients
Velocity gradients
Step gradients
2. Precipitation
ammonium sulfate, PEG and sodium chloride
3. Ultrafiltration
4. column chromatography
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.
Velocity gradients
• the virus moves at a rate determined by its
sedimentation coefficient and the density of
the gradient matrix
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.
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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.
Purification by ultracentrifugation
Solute
Saturated solution
w/w (g per 100 g
solution)
Saturated solution
w/v (g per 100 ml
solution)
Density (g cm-3)
Sucrose
67.9
90.9
1.34
Potassium sodium
tartrate
39.71
51.9
1.31
Caesium chloride
65.7
126
1.92
Caesium sulphate
64.5
129.8
2.01
Method
Enrichment
Comments
Low-speed centrifugation
Zero
Essential to remove cellular debris.
Ultracentrifugation
10-500 fold
Commonly used.
Beckman Airfuge®
50 fold
Enrichment from small volumes.
Direct onto grid sedimentation
50-200 fold
Easy to use (e.g. 100 000 g, 30-60 min).
Gradient centrifugation
100-500 fold
Very efficient but time consuming
Sucrose cushion centrifugation
20-200 fold
Time consuming; preserves labile virus
structures
Ultrafiltration: Centricon®
10-100 fold
Expensive; not suitable for labile viruses.
Agar filtration
10 fold
Useful before negative staining.
Molecular sieve
chromatography
Dilution!
Efficient purification.
Precipitation
>100fold
Co-precipitation of contaminants.
Bioaffinity techniques
10-500 fold
Specific antibodies or ligands are required
Solid-phase immuno-EM
10-500 fold
Difficult to establish
Affinity chromatography
10-100 fold
Rarely used.
Step gradients
• Step gradients involve layering a lower
density solution onto a cushion of high
density material.
A discontinuous sucrose density gradient is prepared by layering
successive decreasing sucrose densities solutions upon one another.
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summer 2011
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summer 2011
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Density gradient maker for centrifuge tubes
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summer 2011
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fit the original definition of “….principle of
centrifugal force to separate materials of
different densities,” specifically
• HIGH SPEED centrifuges
aka SUPERSPEED centrifuges
and
• ULTRA centrifuges
ultra = higher. Modern ultras have max speeds
80,000 – 150,000 rpm
up to around
22,000 rpm
High speed centrifuge
•
•
•
•
Beckman Avanti J series
26,000 rpm (revolutions per minute)
82,000 g (gravities)
Weight 600 lb
Ultracentrifuge
•
•
•
•
Beckman OptimaLXP
100,000 rpm
802,400 g
Weight 1025 lb
Mechanical Failure
• Is caused by age and by improper use or
inadequate care of centrifuge or rotor.
Especially the rotor.
Rotors
•A
Fixed
rotor
highangle
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
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.
Vivapure®
Virus Purification and Concentration Kits
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
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
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summer 2011
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summer 2011
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http://www.sciencegateway.org/tools/rotor.htm
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Alfa Wassermann continues ultracentrifugation
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summer 2011
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summer 2011
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summer 2011
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summer 2011
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Acquire Specialty to Take Opportunity
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