purification to obtain the viral particles in a single form out of cell debris i.e. removal of all...
TRANSCRIPT
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
IsopycnicCushion
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
IsoradiationIsoradiation
Infectivity Infectivity
serologicalserological
E.M.E.M.
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiation
Infectivity Infectivity
serologicalserological
E.M.E.M.
MarkingMarking
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiation
Infectivity 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 10
Conc.(mg/ml)
No. of lo
cal le
sio
ns
Virus preparationVirus preparation
y = 1.9608x
020406080
100120140160180
0 2 4 6 8 10
Conc.(mg/ml)
No. of Pla
quesث
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiation
Infectivity Infectivity
serologicalserological
E.M.E.M.
Criteria of purityCriteria of purity
SpectrophotomeSpectrophotometerter
IsoradiationIsoradiation
Infectivity 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)
