28. Collect the flow-through and keep at 4
C.
29. Wash the column with 10 column volumes of binding buffer.
30. Elute the recombinant protein with elution buffer (20 mM
sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4)
and collect several 2-ml fractions.
31. Use an SDS-PAGE gel and western blotting to detect positive
fractions (Fig. 1b).
32. Pool the positive fractions and dialyze four times with PBS.
33. Measure the recombinant protein concentration and store
small aliquots at À80
C.
3.2 Vaccination,
Immune Responses,
and Dual Challenge
3.2.1 Immunization
of Mice and Serum
Collection
1. BALB/c and A/J wild-type mice as well as C57BL/6
CD4-knockout (KO) and CD8-KO (6–8 week) mice are
weighed and anaesthetized with 100 μl of 80 mg/kg ketamine
and 6 mg/kg xylazine via intraperitoneal injection.
2. After 5 min of anesthetic injection, the mice receive 10 μl of a
mixture containing 30 μg of chimeric vaccine candidate plus
60 μg of PA (see Note 1, Fig. 2) in each nostril. The control
group receives only 60 μg of PA (see Note 2).
3. Mice are immunized at 0, 2, and 5 weeks and bled just before
each immunization and on week 7 via the submandibular
method with 5-mm lancets.
4. Centrifuge the blood sample at 13,000 Â g for 10 min. at 4
C,
collect the sera, and keep at À20
C until use.
3.2.2 ELISA
Antigen-specific antibody responses are measured in the sera of
naive and immunized mice by ELISA.
1. Ninety-six-well Corning EIA/RIA plates are coated with 1 μg/
ml of recombinant antigens (EFn-3ÂM2e-HA2, 3ÂM2eHA2, and PR8) at 4
C overnight.
2. Wash five times the plates with PBS-T (PBS with 0.05% Tween20) using an ImmunoWash™ 1575 microplate washer
(Bio-Rad, Hercules, CA).
3. Block the wells with 200 μl of 1% bovine serum albumin (BSA)
in PBS for 1 h at room temperature.
4. Dilute the serum samples 1:100, add them to the 96-well plate,
and incubate for 2 h at room temperature with rocking.
5. Standard plates are coated with capture antibodies diluted
1:500 in PBS-T, including affinity-purified anti-mouse
IgG-Fc, IgG1, IgG2a, or IgA (Bethyl Laboratories Inc.,
Montgomery, TX).
6. Repeat step 2.
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Diana Diaz-Are ´ valo et al.
C.
29. Wash the column with 10 column volumes of binding buffer.
30. Elute the recombinant protein with elution buffer (20 mM
sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4)
and collect several 2-ml fractions.
31. Use an SDS-PAGE gel and western blotting to detect positive
fractions (Fig. 1b).
32. Pool the positive fractions and dialyze four times with PBS.
33. Measure the recombinant protein concentration and store
small aliquots at À80
C.
3.2 Vaccination,
Immune Responses,
and Dual Challenge
3.2.1 Immunization
of Mice and Serum
Collection
1. BALB/c and A/J wild-type mice as well as C57BL/6
CD4-knockout (KO) and CD8-KO (6–8 week) mice are
weighed and anaesthetized with 100 μl of 80 mg/kg ketamine
and 6 mg/kg xylazine via intraperitoneal injection.
2. After 5 min of anesthetic injection, the mice receive 10 μl of a
mixture containing 30 μg of chimeric vaccine candidate plus
60 μg of PA (see Note 1, Fig. 2) in each nostril. The control
group receives only 60 μg of PA (see Note 2).
3. Mice are immunized at 0, 2, and 5 weeks and bled just before
each immunization and on week 7 via the submandibular
method with 5-mm lancets.
4. Centrifuge the blood sample at 13,000 Â g for 10 min. at 4
C,
collect the sera, and keep at À20
C until use.
3.2.2 ELISA
Antigen-specific antibody responses are measured in the sera of
naive and immunized mice by ELISA.
1. Ninety-six-well Corning EIA/RIA plates are coated with 1 μg/
ml of recombinant antigens (EFn-3ÂM2e-HA2, 3ÂM2eHA2, and PR8) at 4
C overnight.
2. Wash five times the plates with PBS-T (PBS with 0.05% Tween20) using an ImmunoWash™ 1575 microplate washer
(Bio-Rad, Hercules, CA).
3. Block the wells with 200 μl of 1% bovine serum albumin (BSA)
in PBS for 1 h at room temperature.
4. Dilute the serum samples 1:100, add them to the 96-well plate,
and incubate for 2 h at room temperature with rocking.
5. Standard plates are coated with capture antibodies diluted
1:500 in PBS-T, including affinity-purified anti-mouse
IgG-Fc, IgG1, IgG2a, or IgA (Bethyl Laboratories Inc.,
Montgomery, TX).
6. Repeat step 2.
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Diana Diaz-Are ´ valo et al.