3.5 Characterization
of Liposome Size,
Shape, and Charge
1. Dynamic light scattering on a Zetasizer Nano ZS90 (Malvern)
was used to evaluate the particle diameter and zeta potential of
liposomes at 25
C. All experiments were conducted using a
4-mW, 633-nm HeNe laser as the light source at a fixed measuring angle of 90
to the incident laser beam.
2. Images of the LEPS particles were obtained through JEOL
JSM-CXII transmission electron microscopy analysis at
100 kV, with samples prepared by dip-coating a 200-mesh
formvar and a carbon-coated grid (FCF-200-Cu-TB, Electron
Microscopy Sciences), followed by negative staining using a 1%
solution of uranyl acetate.
3.6 Murine
Immunization
with LEPS
1. Following assembly and characterization, the LEPS vaccine can
then be used for animal immunizations. Standardize dosage of
LEPS to include 2.2 μg of the 19F polysaccharide.
2. Prior to the first immunization, collect serum from the mice via
retro-orbital bleeding followed by centrifugation to establish
baseline antibody and cytokine levels.
3. Immunize mice (female CD-1 mice) in groups of 6 using
subcutaneous (SC) injections of 200 μL. Include a sham vaccination group that are immunized with PBS as a negative
control.
4. After the passage of 14 days, collect serum from the mice as
described above to measure antibody and cytokine values postprimary immunization.
5. After collecting the post-primary immunization serum, immunize the mice a second time using the same conditions.
6. After the passage of 14 days, collect serum from the mice as
described above to measure antibody and cytokine values postsecondary immunization.
3.7 Quantification
of the Immune
Response
1. To characterize relative antibody titers associated with delivery
optimization, perform an enzyme-linked immunosorbent assay
(ELISA). First, coat a 96-well Costar high-binding polystyrene
plate with polysaccharide 19F (10 μg/mL) in tris-buffered
saline (TBS) at 4
C overnight.
2. Next, block the plate with 3% bovine serum albumin in TBS–
Tween 20 (TBS-T) for 1 h at 22
C.
3. Add sera that has been diluted into TBS-T in ratios of 1:1000,
1:5000, 1:7500, and 1:10,000 to the plate.
4. Incubate the plates at 37
C with mild agitation for 3 h.
5. Add the secondary antibody [anti-mouse IgG, IgA, IgM
(H + L), IgE, highly X-adsorbed (biotin)] to the wells in a
1:1000 ratio and agitate for 2 h.
Liposomal Dual Antigen Delivery
485
of Liposome Size,
Shape, and Charge
1. Dynamic light scattering on a Zetasizer Nano ZS90 (Malvern)
was used to evaluate the particle diameter and zeta potential of
liposomes at 25
C. All experiments were conducted using a
4-mW, 633-nm HeNe laser as the light source at a fixed measuring angle of 90
to the incident laser beam.
2. Images of the LEPS particles were obtained through JEOL
JSM-CXII transmission electron microscopy analysis at
100 kV, with samples prepared by dip-coating a 200-mesh
formvar and a carbon-coated grid (FCF-200-Cu-TB, Electron
Microscopy Sciences), followed by negative staining using a 1%
solution of uranyl acetate.
3.6 Murine
Immunization
with LEPS
1. Following assembly and characterization, the LEPS vaccine can
then be used for animal immunizations. Standardize dosage of
LEPS to include 2.2 μg of the 19F polysaccharide.
2. Prior to the first immunization, collect serum from the mice via
retro-orbital bleeding followed by centrifugation to establish
baseline antibody and cytokine levels.
3. Immunize mice (female CD-1 mice) in groups of 6 using
subcutaneous (SC) injections of 200 μL. Include a sham vaccination group that are immunized with PBS as a negative
control.
4. After the passage of 14 days, collect serum from the mice as
described above to measure antibody and cytokine values postprimary immunization.
5. After collecting the post-primary immunization serum, immunize the mice a second time using the same conditions.
6. After the passage of 14 days, collect serum from the mice as
described above to measure antibody and cytokine values postsecondary immunization.
3.7 Quantification
of the Immune
Response
1. To characterize relative antibody titers associated with delivery
optimization, perform an enzyme-linked immunosorbent assay
(ELISA). First, coat a 96-well Costar high-binding polystyrene
plate with polysaccharide 19F (10 μg/mL) in tris-buffered
saline (TBS) at 4
C overnight.
2. Next, block the plate with 3% bovine serum albumin in TBS–
Tween 20 (TBS-T) for 1 h at 22
C.
3. Add sera that has been diluted into TBS-T in ratios of 1:1000,
1:5000, 1:7500, and 1:10,000 to the plate.
4. Incubate the plates at 37
C with mild agitation for 3 h.
5. Add the secondary antibody [anti-mouse IgG, IgA, IgM
(H + L), IgE, highly X-adsorbed (biotin)] to the wells in a
1:1000 ratio and agitate for 2 h.
Liposomal Dual Antigen Delivery
485