6. Allow at least 4 weeks before bleeding the mice for immune
sera. This time is required for isotype-switching of the antibodies from IgM to IgG (see Note 3).
3.3 Hyperimmune
Sera Production
1. For intranasal colonization of S. pneumoniae thaw a frozen
bacterial aliquot (Subheading 3.1) on ice.
2. Centrifuge at 1700 rcf for 5 min at room temperature (RT),
discard the supernatant and wash once with 1 ml of sterile PBS
to get rid of the glycerol-containing media.
3. Resuspend and dilute the bacterial pellet in sterile PBS to
achieve 5 Â 10
6 CFU/10 μl and proceed with intranasal challenge in mice (see Note 4).
4. Plate the bacterial suspension on blood agar to confirm the
dosage. Perform the inoculation three times, each 7 days apart.
5. A week after three intranasal S. pneumoniae inoculations, vaccinate mice with Prevnar 13
® as in Subheading 3.2.
6. Four weeks after Prevnar 13
® , euthanize mice and collect
hyperimmune sera (see Note 5).
3.4 Sera Collection
and Storage
1. Euthanize the mouse using CO 2 euthanasia. Immediately,
spray ethanol on the chest area and insert a 1.0 ml syringe
with 25-G needle into the heart and draw blood. Alternatively,
snip the portal vein and collect the blood using a pipette (see
Note 6). Transfer the blood to Microtainer tubes.
2. Immediately, spin the Microtainer tubes for 2 min at 7607 rcf
at room temperature in a tabletop centrifuge.
3. Transfer the sera (the clear part on top of the gel) to a sterile
15 ml falcon tube to pool the sera from all the mice.
4. Aliquot in small volumes of 50–100 μl in microcentrifuge tubes
to avoid repeated freeze-thawing of large volumes of
immune sera.
5. Save at À80
C until ready to use (see Note 7).
3.5 Quantification
of Antibodies Bound
to the Bacterial
Surface by Flow
Cytometry
1. Thaw an aliquot of the S. pneumoniae TIGR4 strain. If available, use a bacterial strain expressing GFP-tag (see Note 8).
2. Once the aliquot of S. pneumoniae is thawed, spin the tube at
1700 rcf for 5 min at room temperature. Discard the supernatant. Resuspend the pellet in +++ buffer (similar buffer used for
OPH assays) at a final concentration of 4 Â 10
7 CFU/ml. Use
25 μl per well so as to have 10
6 bacteria per reaction. The
amount of bacteria is important to balance feasible detection
without diluting the antibodies present in the immune sera.
3. Add the 25 μl of diluted bacteria to the appropriate wells of a
round bottom 96-well plate. Use different volumes of
immune-sera to opsonize the bacteria. Use +++ buffer to
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Manmeet Bhalla et al.
sera. This time is required for isotype-switching of the antibodies from IgM to IgG (see Note 3).
3.3 Hyperimmune
Sera Production
1. For intranasal colonization of S. pneumoniae thaw a frozen
bacterial aliquot (Subheading 3.1) on ice.
2. Centrifuge at 1700 rcf for 5 min at room temperature (RT),
discard the supernatant and wash once with 1 ml of sterile PBS
to get rid of the glycerol-containing media.
3. Resuspend and dilute the bacterial pellet in sterile PBS to
achieve 5 Â 10
6 CFU/10 μl and proceed with intranasal challenge in mice (see Note 4).
4. Plate the bacterial suspension on blood agar to confirm the
dosage. Perform the inoculation three times, each 7 days apart.
5. A week after three intranasal S. pneumoniae inoculations, vaccinate mice with Prevnar 13
® as in Subheading 3.2.
6. Four weeks after Prevnar 13
® , euthanize mice and collect
hyperimmune sera (see Note 5).
3.4 Sera Collection
and Storage
1. Euthanize the mouse using CO 2 euthanasia. Immediately,
spray ethanol on the chest area and insert a 1.0 ml syringe
with 25-G needle into the heart and draw blood. Alternatively,
snip the portal vein and collect the blood using a pipette (see
Note 6). Transfer the blood to Microtainer tubes.
2. Immediately, spin the Microtainer tubes for 2 min at 7607 rcf
at room temperature in a tabletop centrifuge.
3. Transfer the sera (the clear part on top of the gel) to a sterile
15 ml falcon tube to pool the sera from all the mice.
4. Aliquot in small volumes of 50–100 μl in microcentrifuge tubes
to avoid repeated freeze-thawing of large volumes of
immune sera.
5. Save at À80
C until ready to use (see Note 7).
3.5 Quantification
of Antibodies Bound
to the Bacterial
Surface by Flow
Cytometry
1. Thaw an aliquot of the S. pneumoniae TIGR4 strain. If available, use a bacterial strain expressing GFP-tag (see Note 8).
2. Once the aliquot of S. pneumoniae is thawed, spin the tube at
1700 rcf for 5 min at room temperature. Discard the supernatant. Resuspend the pellet in +++ buffer (similar buffer used for
OPH assays) at a final concentration of 4 Â 10
7 CFU/ml. Use
25 μl per well so as to have 10
6 bacteria per reaction. The
amount of bacteria is important to balance feasible detection
without diluting the antibodies present in the immune sera.
3. Add the 25 μl of diluted bacteria to the appropriate wells of a
round bottom 96-well plate. Use different volumes of
immune-sera to opsonize the bacteria. Use +++ buffer to
564
Manmeet Bhalla et al.