6. Then the electroporation is applied: three 50 ms long pulses at
100 V (200 V/cm) followed by three more pulses of the
opposite polarity delivered to the injection site.
7. Then atipamezole (1 mg/kg) is injected to reverse the effect of
anesthetics. Animals start to recover after 10 min (see Note 5).
8. At week 2, a second immunization is performed as above.
9. At week 4 and 6, two protein boosts alone or combined with
adjuvant are given by intramuscular injection.
10. At week 3, 5 and 7, blood samples are collected by cheek
bleeding, for collection of sera for the different immune
response assays.
11. At week 8, half of the mice in each group will be anesthetized
and cardiac bleeding is performed to get a maximum amount
of sera for neutralization antibody assays.
12. Then ELISA and plaque-reduction neutralization test (PRNT)
are performed to evaluate antibody responses.
13. Bones and spleens can be collected to measure cellular immunity by ELISPOT assay.
14. The remaining mice can be challenged by IP injection of virus
(see Note 6).
3.3 Antibody Assay
by ELISA
1. Dilute capture antigen in Coating Buffer to 1 μg/mL. 10 mL is
required per ELISA plate (see Note 7).
2. Add 100 μL/well of the diluted antigen using a multichannel
pipette.
3. Ensure the solution is distributed over entire surface of all
wells.
4. Cover the plate with a plastic seal.
5. Incubate overnight at 2–8
C.
6. Flick off contents of microplate into the sink and wash plates
four times with Washing Buffer >250 μL/well (see Note 8).
7. Dry all surfaces of plates with a paper towel.
8. Add 200 μL/well of Blocking Buffer.
9. Cover the plate with a plastic seal.
10. Incubate for at least 30 min at RT.
11. Dilute samples serially in Blocking Buffer in a 96-well U-bottom plate. Always prepare enough volume for a duplicate set
per sample as 100 μL/well of samples are required.
DNA Vaccine Design and Adjuvant Selection
415
100 V (200 V/cm) followed by three more pulses of the
opposite polarity delivered to the injection site.
7. Then atipamezole (1 mg/kg) is injected to reverse the effect of
anesthetics. Animals start to recover after 10 min (see Note 5).
8. At week 2, a second immunization is performed as above.
9. At week 4 and 6, two protein boosts alone or combined with
adjuvant are given by intramuscular injection.
10. At week 3, 5 and 7, blood samples are collected by cheek
bleeding, for collection of sera for the different immune
response assays.
11. At week 8, half of the mice in each group will be anesthetized
and cardiac bleeding is performed to get a maximum amount
of sera for neutralization antibody assays.
12. Then ELISA and plaque-reduction neutralization test (PRNT)
are performed to evaluate antibody responses.
13. Bones and spleens can be collected to measure cellular immunity by ELISPOT assay.
14. The remaining mice can be challenged by IP injection of virus
(see Note 6).
3.3 Antibody Assay
by ELISA
1. Dilute capture antigen in Coating Buffer to 1 μg/mL. 10 mL is
required per ELISA plate (see Note 7).
2. Add 100 μL/well of the diluted antigen using a multichannel
pipette.
3. Ensure the solution is distributed over entire surface of all
wells.
4. Cover the plate with a plastic seal.
5. Incubate overnight at 2–8
C.
6. Flick off contents of microplate into the sink and wash plates
four times with Washing Buffer >250 μL/well (see Note 8).
7. Dry all surfaces of plates with a paper towel.
8. Add 200 μL/well of Blocking Buffer.
9. Cover the plate with a plastic seal.
10. Incubate for at least 30 min at RT.
11. Dilute samples serially in Blocking Buffer in a 96-well U-bottom plate. Always prepare enough volume for a duplicate set
per sample as 100 μL/well of samples are required.
DNA Vaccine Design and Adjuvant Selection
415