2. Virus stock solution is added (9 mL for SW 40 Ti or 8 mL for
SW 41 Ti) onto the sucrose gradient and centrifugated at
160,000 Â g (30,000 rpm in SW 40 Ti or SW41 Ti rotor) for
90 min at +4
C.
3. Virus settled near the interface between the 20% and 50%
sucrose layers is collected by discarding the medium fraction
and the bottom 0.8 mL consisting of 50% sucrose.
3.10.2 Centriprep
Concentration
1. Virus stocks are loaded onto the sample container of the Centriprep concentrator as described by the manufacturer.
2. The assembled concentrator is centrifugated at an appropriate
g-force (according to the manufacturer’s recommendations),
until the fluid levels inside and outside the filtrate collector
equilibrate.
3. The device is removed, the airtight seal cap is snapped off, the
filtrate is decanted, the cap is replaced, and the concentrator is
centrifugated a second time.
4. The filtrate is decanted, the twist-lock cap is loosened, and the
filtrate collector is removed.
5. The concentrated virus sample is collected with a 1 mL disposable plastic pipette. If further concentration of virus is desired,
it can be centrifugated again after decanting the filtrate.
3.10.3 Affinity
Chromatography
Concentration
Efficient removal of endotoxins and other contaminants can be
achieved by application of Matrex
® Cellufine™ Sulfate columns,
which also provides a convenient procedure for virus stock concentration. The manufacturer’s recommendations should be followed
as described below.
1. The affinity matrix column is equilibrated with adsorption
buffer (0.01 M phosphate, 0.1 M NaCl, pH 7.5) and samples
are loaded at pH 7.5.
2. The column is washed with several bed volumes of adsorption
buffer to remove nonbinding contaminants and the concentrated virus is eluted with elution buffer (1–2 M NaCl or KCl).
3.11 Immunizations
Although the topic of this chapter relates to antigen preparation it is
essential to include some information of the immunization process
for RNA, viral particles, and DNA. In the context of alphavirusbased vaccine development, a large number of immunization studies have indeed been conducted [5].
3.11.1 Immunization
of Mice with RNA
In vitro–transcribed alphavirus RNA can be directly used for
administration of RNA replicons for immunization experiments in
mice, as follows [20].
76
Kenneth Lundstrom
SW 41 Ti) onto the sucrose gradient and centrifugated at
160,000 Â g (30,000 rpm in SW 40 Ti or SW41 Ti rotor) for
90 min at +4
C.
3. Virus settled near the interface between the 20% and 50%
sucrose layers is collected by discarding the medium fraction
and the bottom 0.8 mL consisting of 50% sucrose.
3.10.2 Centriprep
Concentration
1. Virus stocks are loaded onto the sample container of the Centriprep concentrator as described by the manufacturer.
2. The assembled concentrator is centrifugated at an appropriate
g-force (according to the manufacturer’s recommendations),
until the fluid levels inside and outside the filtrate collector
equilibrate.
3. The device is removed, the airtight seal cap is snapped off, the
filtrate is decanted, the cap is replaced, and the concentrator is
centrifugated a second time.
4. The filtrate is decanted, the twist-lock cap is loosened, and the
filtrate collector is removed.
5. The concentrated virus sample is collected with a 1 mL disposable plastic pipette. If further concentration of virus is desired,
it can be centrifugated again after decanting the filtrate.
3.10.3 Affinity
Chromatography
Concentration
Efficient removal of endotoxins and other contaminants can be
achieved by application of Matrex
® Cellufine™ Sulfate columns,
which also provides a convenient procedure for virus stock concentration. The manufacturer’s recommendations should be followed
as described below.
1. The affinity matrix column is equilibrated with adsorption
buffer (0.01 M phosphate, 0.1 M NaCl, pH 7.5) and samples
are loaded at pH 7.5.
2. The column is washed with several bed volumes of adsorption
buffer to remove nonbinding contaminants and the concentrated virus is eluted with elution buffer (1–2 M NaCl or KCl).
3.11 Immunizations
Although the topic of this chapter relates to antigen preparation it is
essential to include some information of the immunization process
for RNA, viral particles, and DNA. In the context of alphavirusbased vaccine development, a large number of immunization studies have indeed been conducted [5].
3.11.1 Immunization
of Mice with RNA
In vitro–transcribed alphavirus RNA can be directly used for
administration of RNA replicons for immunization experiments in
mice, as follows [20].
76
Kenneth Lundstrom