25. The MOI can be increased to 1.0 and efficient killing will still
occur. However, high MOI with virulent S. pneumoniae strains
like TIGR4 should be avoided because of the risk of neutrophil
death due to the release of the pore-forming toxin pneumolysin by the bacteria [20]. We recommend that the MOI be
adjusted depending upon the bacterial strain used.
26. Do not exceed the opsonization incubation more than 30 min.
If kept longer, the bacteria could start replicating and this
could affect the MOI. Alternatively, you can skip the preopsonization over time step and mix the sera and bacteria, then
immediately proceed to adding the mix to the PMNs.
27. An alternative approach when comparing OPH efficacy of the
sera is to test a range of percentages as for the bacterial binding
assay. We found a dose dependent increase in bacterial killing
by PMNs when the sera were increased from 1% to 3% to 10%.
However, we found that adding more sera did not increase
bacterial killing and in fact using >10% sera triggered bacterial
growth and impaired their killing by PMNs.
28. Add equal volume of 40% glycerol in THY broth to each well
and store the plate at À20
C. This is helpful in case there is a
need to replate the reaction conditions for any reason (e.g.,
bacterial CFU too high to be counted accurately).
29. These assay parameters allow for the differentiation in opsonophagocytic capacity of different immune vs. naive sera (Fig. 3).
This assay, when using the same opsonic conditions, further
allows the testing of intrinsic PMN function. For example,
PMNs from old mice have an intrinsic decline in antibacterial
function as they fail to kill S. pneumoniae as efficiently as PMNs
from young counterparts, even when the bacteria are opsonized with same sera (Fig. 3).
30. Hyperimmune sera can be used as “in-house” standard reference sera for the OPH assays as well as the antibody bacterial
binding assays. Both bacterial killing and antibody binding
obtained by using hyperimmune sera can be set to 1%
(or 100%) and can then be used to compare the opsonophagocytic and bacterial binding efficiency of the test-sera.
31. The same assay can be used with PMNs isolated from any other
source such as tissue or circulating PMNs [21].
Acknowledgments
This work was supported by National Institute of Health grant
R00AG051784 to E.N.B.G.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
573
occur. However, high MOI with virulent S. pneumoniae strains
like TIGR4 should be avoided because of the risk of neutrophil
death due to the release of the pore-forming toxin pneumolysin by the bacteria [20]. We recommend that the MOI be
adjusted depending upon the bacterial strain used.
26. Do not exceed the opsonization incubation more than 30 min.
If kept longer, the bacteria could start replicating and this
could affect the MOI. Alternatively, you can skip the preopsonization over time step and mix the sera and bacteria, then
immediately proceed to adding the mix to the PMNs.
27. An alternative approach when comparing OPH efficacy of the
sera is to test a range of percentages as for the bacterial binding
assay. We found a dose dependent increase in bacterial killing
by PMNs when the sera were increased from 1% to 3% to 10%.
However, we found that adding more sera did not increase
bacterial killing and in fact using >10% sera triggered bacterial
growth and impaired their killing by PMNs.
28. Add equal volume of 40% glycerol in THY broth to each well
and store the plate at À20
C. This is helpful in case there is a
need to replate the reaction conditions for any reason (e.g.,
bacterial CFU too high to be counted accurately).
29. These assay parameters allow for the differentiation in opsonophagocytic capacity of different immune vs. naive sera (Fig. 3).
This assay, when using the same opsonic conditions, further
allows the testing of intrinsic PMN function. For example,
PMNs from old mice have an intrinsic decline in antibacterial
function as they fail to kill S. pneumoniae as efficiently as PMNs
from young counterparts, even when the bacteria are opsonized with same sera (Fig. 3).
30. Hyperimmune sera can be used as “in-house” standard reference sera for the OPH assays as well as the antibody bacterial
binding assays. Both bacterial killing and antibody binding
obtained by using hyperimmune sera can be set to 1%
(or 100%) and can then be used to compare the opsonophagocytic and bacterial binding efficiency of the test-sera.
31. The same assay can be used with PMNs isolated from any other
source such as tissue or circulating PMNs [21].
Acknowledgments
This work was supported by National Institute of Health grant
R00AG051784 to E.N.B.G.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
573