6. Add streptavidin conjugated with alkaline phosphatase to each
well in a 1:1000 ratio and shake for 30 min.
7. Develop the signal with p-nitrophenylphosphate, and quench
the reaction using 0.75 M NaOH.
8. Measure the absorbance at 405 nm for each well using a plate
reader spectrophotometer. Relative antibody titers should be
determined through comparison to the sham vaccine control.
9. Cytokine measurements should be determined using ELISA
kits (R&D Systems) following the manufacturer’s instructions.
4 Notes
1. The 0.6 mg/mL concentration is when liposomes with the
current lipid composition show the maximum encapsulation
efficiency for polysaccharide 19F [5].
2. Large vesicles, due to several reasons such as multilamellarity
and small core volume, are not ideal candidates for the purpose
of the study [11, 12]. By extruding the large multilamellar
vesicles with size of around 1–5 μm, while passing through
the polycarbonate membrane, their lipid bilayers disrupt and
immediately reform to unilamellar vesicles with a size distribution related to the membrane pore size (which, here, is
200 nm), exit the membrane, and result in liposomes with
defined size on the other side with more stability and improved
encapsulation efficiency (due to a larger core volume).
3. The GFP that has been purified from recombinant E. coli must
be evaluated for purity and concentration (in our case, by using
SDS-PAGE and Bradford analysis, respectively).
4. To protect the sealed microplate during the polysaccharide
detection assay, it is placed in a small, sealable plastic bag
prior to addition to the surface of a 92
C water bath.
5. Considering differences in monosaccharide compositions of
polysaccharides [13, 14], it is necessary to optimize the colorimetric assay to find the maximum absorbance wavelength for
each polysaccharide. In this case, the corresponding peak for
polysaccharide 19F was observed at 480 nm.
References
1. Black RE, Cousens S, Johnson HL, Lawn JE,
Rudan I, Bassani DG, Jha P, Campbell H,
Walker CF, Cibulskis R, Eisele T, Liu L,
Mathers C, Child Health Epidemiology Reference Group of WHO, Unicef (2010) Global,
regional, and national causes of child mortality
in 2008: a systematic analysis. Lancet 375
(9730):1969–1987.
https://doi.org/10.
1016/S0140-6736(10)60549-1
2. Vergison A, Dagan R, Arguedas A,
Bonhoeffer J, Cohen R, Dhooge I,
Hoberman A, Liese J, Marchisio P, Palmu AA,
Ray GT, Sanders EA, Simoes EA, Uhari M, van
Eldere J, Pelton SI (2010) Otitis media and its
486
Roozbeh Nayerhoda et al.
well in a 1:1000 ratio and shake for 30 min.
7. Develop the signal with p-nitrophenylphosphate, and quench
the reaction using 0.75 M NaOH.
8. Measure the absorbance at 405 nm for each well using a plate
reader spectrophotometer. Relative antibody titers should be
determined through comparison to the sham vaccine control.
9. Cytokine measurements should be determined using ELISA
kits (R&D Systems) following the manufacturer’s instructions.
4 Notes
1. The 0.6 mg/mL concentration is when liposomes with the
current lipid composition show the maximum encapsulation
efficiency for polysaccharide 19F [5].
2. Large vesicles, due to several reasons such as multilamellarity
and small core volume, are not ideal candidates for the purpose
of the study [11, 12]. By extruding the large multilamellar
vesicles with size of around 1–5 μm, while passing through
the polycarbonate membrane, their lipid bilayers disrupt and
immediately reform to unilamellar vesicles with a size distribution related to the membrane pore size (which, here, is
200 nm), exit the membrane, and result in liposomes with
defined size on the other side with more stability and improved
encapsulation efficiency (due to a larger core volume).
3. The GFP that has been purified from recombinant E. coli must
be evaluated for purity and concentration (in our case, by using
SDS-PAGE and Bradford analysis, respectively).
4. To protect the sealed microplate during the polysaccharide
detection assay, it is placed in a small, sealable plastic bag
prior to addition to the surface of a 92
C water bath.
5. Considering differences in monosaccharide compositions of
polysaccharides [13, 14], it is necessary to optimize the colorimetric assay to find the maximum absorbance wavelength for
each polysaccharide. In this case, the corresponding peak for
polysaccharide 19F was observed at 480 nm.
References
1. Black RE, Cousens S, Johnson HL, Lawn JE,
Rudan I, Bassani DG, Jha P, Campbell H,
Walker CF, Cibulskis R, Eisele T, Liu L,
Mathers C, Child Health Epidemiology Reference Group of WHO, Unicef (2010) Global,
regional, and national causes of child mortality
in 2008: a systematic analysis. Lancet 375
(9730):1969–1987.
https://doi.org/10.
1016/S0140-6736(10)60549-1
2. Vergison A, Dagan R, Arguedas A,
Bonhoeffer J, Cohen R, Dhooge I,
Hoberman A, Liese J, Marchisio P, Palmu AA,
Ray GT, Sanders EA, Simoes EA, Uhari M, van
Eldere J, Pelton SI (2010) Otitis media and its
486
Roozbeh Nayerhoda et al.