3 Methods
This section has further been divided into subsections to include
the outlined techniques for evaluating the vaccine-induced antibody response in vitro. These include culturing bacteria, immunizing mice, collecting and storing immune sera, quantifying antibody
binding to bacterial surfaces using flow cytometry, isolating PMNs
from mouse bone marrow, and OPH assays.
3.1 Growing Bacteria
1. A day prior, streak a blood agar pate with S. pneumoniae and
keep overnight at 37
C in 5% CO 2 .
2. The following day, grow S. pneumoniae at 37
C in 5% CO 2 in
Todd-Hewitt broth supplemented with Oxyrase until the culture reaches mid-exponential phase (OD 0.7–0.8).
3. Centrifuge the culture at 1700 rcf, remove the supernatants
and resuspend in the growth media with 20% (v/v) glycerol.
4. Flash freeze the cells on dry ice and transfer to À80
C for
storage.
5. Prior to use, thaw aliquots, wash and resuspend in PBS to
obtain the appropriate concentration. Confirm the bacterial
number by serial dilution and plating on blood agar plates.
Incubate the plates at 37
C, 5% CO 2 overnight and count
the number of colony forming units (CFU) the next day [10].
3.2 Immune Sera
Production
1. Prevnar 13
® is heat labile and should be kept on ice at all times.
Avoid exposure to high temperature or direct sunlight. Shake
the vaccine vial well to disrupt any visible precipitates (see Note
1).
2. Using an 18 G needle, fill the 1.0 ml syringe with required
amount of vaccine.
3. Discard the 18 G needle and cap the syringe with a 27 G
needle. Place the syringe on ice and proceed with the intramuscular (i.m.) injection immediately.
4. I.m. injections require the assistance of another person. One
individual should scruff the mouse near the ears and extend the
hind leg to stretch it out. The second individual should wipe
the area with a 70% ethanol swab and insert the needle slowly
into the caudal thigh muscle, bevel up. Make sure to insert the
needle pointing toward the tail (not toward the head). This
helps ensure you do not hit the sciatic nerve that runs along the
femur (see Note 2).
5. Slowly inject the 50 μl volume of the vaccine. Remove the
needle gently and observe for any sign of bleeding or leakage.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
563
This section has further been divided into subsections to include
the outlined techniques for evaluating the vaccine-induced antibody response in vitro. These include culturing bacteria, immunizing mice, collecting and storing immune sera, quantifying antibody
binding to bacterial surfaces using flow cytometry, isolating PMNs
from mouse bone marrow, and OPH assays.
3.1 Growing Bacteria
1. A day prior, streak a blood agar pate with S. pneumoniae and
keep overnight at 37
C in 5% CO 2 .
2. The following day, grow S. pneumoniae at 37
C in 5% CO 2 in
Todd-Hewitt broth supplemented with Oxyrase until the culture reaches mid-exponential phase (OD 0.7–0.8).
3. Centrifuge the culture at 1700 rcf, remove the supernatants
and resuspend in the growth media with 20% (v/v) glycerol.
4. Flash freeze the cells on dry ice and transfer to À80
C for
storage.
5. Prior to use, thaw aliquots, wash and resuspend in PBS to
obtain the appropriate concentration. Confirm the bacterial
number by serial dilution and plating on blood agar plates.
Incubate the plates at 37
C, 5% CO 2 overnight and count
the number of colony forming units (CFU) the next day [10].
3.2 Immune Sera
Production
1. Prevnar 13
® is heat labile and should be kept on ice at all times.
Avoid exposure to high temperature or direct sunlight. Shake
the vaccine vial well to disrupt any visible precipitates (see Note
1).
2. Using an 18 G needle, fill the 1.0 ml syringe with required
amount of vaccine.
3. Discard the 18 G needle and cap the syringe with a 27 G
needle. Place the syringe on ice and proceed with the intramuscular (i.m.) injection immediately.
4. I.m. injections require the assistance of another person. One
individual should scruff the mouse near the ears and extend the
hind leg to stretch it out. The second individual should wipe
the area with a 70% ethanol swab and insert the needle slowly
into the caudal thigh muscle, bevel up. Make sure to insert the
needle pointing toward the tail (not toward the head). This
helps ensure you do not hit the sciatic nerve that runs along the
femur (see Note 2).
5. Slowly inject the 50 μl volume of the vaccine. Remove the
needle gently and observe for any sign of bleeding or leakage.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
563