(a) Prepare one dose of vaccine containing 125 μg DNA in
final volume of 160 μl (PTAI–DNA ratio 0.8:1 w/w) as
follows:
– Mix 75 μl of prepared PTAI reagent B with 85 μl of
plasmid DNA solution containing 125 μg DNA (see
Subheading 3.3, step 2(a)).
– Incubate for 30 min at RT (20–25
C) with gentle
rocking.
(b) Prepare one dose of vaccine containing 62.5 μg DNA in
final volume of 160 μl (PTAI–DNA ratio—1.6:1 w/w) as
follows:
– Mix 75 μl of prepared PTAI reagent B with 85 μl of
plasmid DNA containing 62.5 μg DNA.
– Incubate for 30 min at RT (20–25
C) with gentle
rocking.
(c) Prepare one dose of vaccine containing 62.5 μg DNA in
final volume of 100 μl (PTAI–DNA ratio—0.8:1 w/w) as
follows:
– Mix 37.5 μl of prepared PTAI reagent B with 62.5 μl of
plasmid DNA solution containing 62.5 μg DNA.
– Incubate for 30 min at RT (20–25
C) with gentle
rocking.
(d) Prepare one dose of vaccine containing 10 μg DNA in
final volume of 50 μl (PTAI–DNA ratio—0.8:1 w/w) as
follows:
– Mix 6.0 μl of prepared PTAI reagent B with 44 μl of
plasmid DNA solution containing 10 μg DNA.
– Incubate for 30 min at RT (20–25
C) with gentle
rocking.
3.4 Carriers Activity
Testing In Vivo
1. Use vaccine mixtures prepared in Subheading 3.3 (steps 2(a)
and 3(a)–(d)) for intramuscular immunization (see Note 12).
2. Collect blood 1 or 2 weeks after each vaccination and use
appropriate test (e.g., ELISA) to evaluate vaccination
effectiveness.
3. Obtain blood sera as follows:
(a) After collection, incubate blood at RT (20–25
C) for 2 h
then at 4
C overnight.
(b) Centrifuge for 10 min at 5000 Â g at 4
C.
(c) Take out the serum and store at 4
C.
4. Evaluate vaccination effectiveness by indirect ELISA assay (see
Note 13).
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