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E. &. O. O. &. Purification of viruses. Purification. To obtain the viral particles in a single form out of cell debris i.e. removal of all impurities. Purification outline. Preparation of harvest. Methods of purification. Criteria of purity. Maintenance of purified virus. - PowerPoint PPT Presentation
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PurificationPurification
To obtain the viral particles in a single form out of cell debris i.e. removal of all impurities.
Purification outlinePurification outline
1.Preparation of harvest.2.Methods of purification.3.Criteria of purity.4.Maintenance of purified
virus.
Preparation of harvest
Bacteria
Plant
Animal Insect
Methods of purificationMethods of purification
1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.
Methods of purificationMethods of purification
1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.
Filtration: :
1.1. UltrafiltrationUltrafiltration 2. Gel filtration2. Gel filtration
Ultra-filtrationUltra-filtration
Gel filtrationGel filtration
Methods of purificationMethods of purification
1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.
centrifugation
Differential
Gradient
Isopycnic Cushion
Methods of purificationMethods of purification
1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.
Adsorption: :
1.1. Ion exchangeIon exchange 2. Heamadsroption2. Heamadsroption
Ion exchange chromatography
HeamadsorptionHeamadsorption
Methods of purificationMethods of purification
1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.
Precipitation: :
1. Chemical1. Chemical• AmmoniumsufateAmmoniumsufate• PEGPEG• Zinc acetaeZinc acetae
2. Physical2. Physical• Isoelectric piont
(pH change)
Methods of purificationMethods of purification
1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.
Methods of purificationMethods of purification
1.Filtration.2.Centrifugation.3.Adsorption.4.Precipitation.5.Solvent extraction.6.Other methods.
Other methodsOther methods
• Electrophoresis.• Serological methods.• Enzymatic reactions.• HPLC
Electrophoresis
HPLCHPLC
Criteria of purityCriteria of purity
Chemical purityChemical purity
Physical purityPhysical purity
Biological purityBiological purity
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiationInfectivity Infectivity
serologicalserological
E.M.E.M.
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiationInfectivity Infectivity
serologicalserological
E.M.E.M.
MarkingMarking
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiationInfectivity Infectivity
serologicalserological
E.M.E.M.
Infectivity::
1. Local lesion assay1. Local lesion assay 2. Plaque assay2. Plaque assay
y = 1.9608x
020406080
100120140160180
0 2 4 6 8 10Conc.(mg/ml)
No. o
f loc
al le
sion
s
Virus preparationVirus preparation
y = 1.9608x
020406080
100120140160180
0 2 4 6 8 10Conc.(mg/ml)
No. o
f Pla
quesث
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiationInfectivity Infectivity
serologicalserological
E.M.E.M.
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiationInfectivity Infectivity
serologicalserological
E.M.E.M.
44 - -MaintenanceMaintenance::
Lyophlization
The half life of infectivity can be measured in seconds at The half life of infectivity can be measured in seconds at 60 60 ooC; minutes at 37 C; minutes at 37 ooC, hours at 20C, hours at 20ooC , days at 4 C , days at 4 ooC and C and years at -70 years at -70 ooC or lower,C or lower, (Fenner (Fenner et alet al, 1987), 1987)
