9. Measuring IgG subtypes is sometimes performed as an indicator of helper T (Th) cell bias, where IgG2 (Th1) and IgG1
(Th2) responses have been associated with a strong cytotoxic T
cell and humoral response, respectively. For example, inclusion
of TLR agonists such as CpG in a vaccine formulation will shift
antibodies to the IgG2 subtype while simultaneously inducing
strong CD8 T cell responses [11]. This is not a hard and fast
rule, as other adjuvants (e.g., sulfated lactosyl archaeol glycolipids) have been shown to induce high levels of antigen-specific
CD8 T cells and predominantly antigen-specific IgG1
antibodies [3].
10. Depending on the vaccine regimen and/or formulation, the
antibody titers in your sample may be too high to measure
accurately with a starting 1:10 dilution. A starting dilution of
1:100 or 1:1000 may be required to properly capture the target
OD in the range of tested serum dilutions. Adjust serum and
dilution buffer volumes in Row A to achieve desired dilution
and then serially dilute as described above. A predilution could
be required to more precisely achieve a 1:1000 starting dilution
(i.e., 1:20 followed by a 1:50).
11. Prior to executing/transferring serial dilutions, it is best to
ensure all tips are tightly attached to the multichannel pipette.
With the large number of repetitive pipetting steps, a tip may
come loose in the middle of the process if not strongly attached
to the pipette.
12. If evaluating IgG subtypes such as IgG2 in mice, it is important
to note the strain of mouse from which the samples were
collected. Commonly used mouse strains such as C57Bl/6
and BALB/c express the IgG2c and IgG2a forms of this antibody, respectively. Keep this in mind when selecting the appropriate secondary antibody for your system.
13. This assay protocol can be adapted to run with other substrates
(e.g., 3,3
0 ,5,5
0 -tetramethylbenzidine) or secondary antibodies
with different enzymatic moieties (e.g., alkaline phosphatase).
However, some optimization will be required to establish a
number of parameters (secondary antibody concentrations,
development time, target OD, etc.). In addition, the plates
will need to be read at the optical density appropriate for the
substrate used.
14. As plates are stacked during incubations, condensation may
form on the underside of some of the plates. Wipe the underside of the plate to remove any condensation prior to loading in
the spectrophotometer as the water droplets may interfere with
the absorbance readings.
15. The selection of an appropriate target OD will depend on the
background and peak signals observed in your assay. We
Determination of Antigen-Specific IgG Antibody Titers
545
(Th2) responses have been associated with a strong cytotoxic T
cell and humoral response, respectively. For example, inclusion
of TLR agonists such as CpG in a vaccine formulation will shift
antibodies to the IgG2 subtype while simultaneously inducing
strong CD8 T cell responses [11]. This is not a hard and fast
rule, as other adjuvants (e.g., sulfated lactosyl archaeol glycolipids) have been shown to induce high levels of antigen-specific
CD8 T cells and predominantly antigen-specific IgG1
antibodies [3].
10. Depending on the vaccine regimen and/or formulation, the
antibody titers in your sample may be too high to measure
accurately with a starting 1:10 dilution. A starting dilution of
1:100 or 1:1000 may be required to properly capture the target
OD in the range of tested serum dilutions. Adjust serum and
dilution buffer volumes in Row A to achieve desired dilution
and then serially dilute as described above. A predilution could
be required to more precisely achieve a 1:1000 starting dilution
(i.e., 1:20 followed by a 1:50).
11. Prior to executing/transferring serial dilutions, it is best to
ensure all tips are tightly attached to the multichannel pipette.
With the large number of repetitive pipetting steps, a tip may
come loose in the middle of the process if not strongly attached
to the pipette.
12. If evaluating IgG subtypes such as IgG2 in mice, it is important
to note the strain of mouse from which the samples were
collected. Commonly used mouse strains such as C57Bl/6
and BALB/c express the IgG2c and IgG2a forms of this antibody, respectively. Keep this in mind when selecting the appropriate secondary antibody for your system.
13. This assay protocol can be adapted to run with other substrates
(e.g., 3,3
0 ,5,5
0 -tetramethylbenzidine) or secondary antibodies
with different enzymatic moieties (e.g., alkaline phosphatase).
However, some optimization will be required to establish a
number of parameters (secondary antibody concentrations,
development time, target OD, etc.). In addition, the plates
will need to be read at the optical density appropriate for the
substrate used.
14. As plates are stacked during incubations, condensation may
form on the underside of some of the plates. Wipe the underside of the plate to remove any condensation prior to loading in
the spectrophotometer as the water droplets may interfere with
the absorbance readings.
15. The selection of an appropriate target OD will depend on the
background and peak signals observed in your assay. We
Determination of Antigen-Specific IgG Antibody Titers
545