2. Add 200 μL of Blocking Buffer to each well.
3. Stack plates and cover with plastic wrap and incubate for 1 h at
37
C.
3.4 Preparation
of Sample Dilutions
1. Centrifuge samples briefly to separate any potential solid debris
from sample. If samples are in a 96-well cluster tube box, spin
samples briefly at 500 Â g for 5 min in the swinging bucket
centrifuge, using the plate rotor. If samples are stored in microcentrifuge tubes, spin for 1 min at 10,000 Â g in
microcentrifuge.
2. For each set of ten samples, one dilution plate (either a nontreated U-bottom 96-well disposable plate or 0.5 mL deepwell assay block plate, depending on required sample volume)
will be loaded with the appropriate volume of diluent buffer
and be used to perform a serial dilution of each serum sample.
Eight data points are generated per sample using a half log
serial dilution to enable the determination of antibody titer
(Fig. 2).
3. The required sample dilution volumes will depend on the
number of antigens or antibody isotypes that will be tested
simultaneously. While routine testing may include a single
antibody isotype (e.g., IgG), multiple isotypes (e.g., IgG,
IgG1, and IgG2a) may be tested in parallel, for example to
evaluate the Th1 vs. Th2 bias of the humoral response (see
Note 9). Alternatively, a single sample’s ability to cross-react
with multiple viral serotypes can be evaluated on separate
plates.
4. For a single antibody isotype–antigen combination, pipette
150 μL of dilution buffer in rows B-H of a U-bottom 96-well
disposable plate. For row A, 180 μL of dilution buffer is added
to each well. Add 20 μL of the test serum sample to a well in
Row A to create a starting 1:10 dilution (see Note 10). If
available, place positive control (e.g., serum from previously
tested animal) in well A11 and negative control in well A12
(e.g., serum from nonimmunized animal) and dilute as above.
Once all samples have been loaded, use a multichannel to
serially dilute 69.4 μL of diluted serum sample 7 times between
rows B-H by pipetting up and down 4–6 times (see Note 11).
5. If running three plates per sample, use a 0.5 mL deep-well assay
block plate and add 400 and 540 μL of dilution buffer to rows
B-H and A, respectively. 60 μL of the test serum would be
added to Row A to create a starting 1:10 dilution and the serial
dilutions would be performed by pipetting 185 μL of sample
down the rows.
Determination of Antigen-Specific IgG Antibody Titers
541
3. Stack plates and cover with plastic wrap and incubate for 1 h at
37
C.
3.4 Preparation
of Sample Dilutions
1. Centrifuge samples briefly to separate any potential solid debris
from sample. If samples are in a 96-well cluster tube box, spin
samples briefly at 500 Â g for 5 min in the swinging bucket
centrifuge, using the plate rotor. If samples are stored in microcentrifuge tubes, spin for 1 min at 10,000 Â g in
microcentrifuge.
2. For each set of ten samples, one dilution plate (either a nontreated U-bottom 96-well disposable plate or 0.5 mL deepwell assay block plate, depending on required sample volume)
will be loaded with the appropriate volume of diluent buffer
and be used to perform a serial dilution of each serum sample.
Eight data points are generated per sample using a half log
serial dilution to enable the determination of antibody titer
(Fig. 2).
3. The required sample dilution volumes will depend on the
number of antigens or antibody isotypes that will be tested
simultaneously. While routine testing may include a single
antibody isotype (e.g., IgG), multiple isotypes (e.g., IgG,
IgG1, and IgG2a) may be tested in parallel, for example to
evaluate the Th1 vs. Th2 bias of the humoral response (see
Note 9). Alternatively, a single sample’s ability to cross-react
with multiple viral serotypes can be evaluated on separate
plates.
4. For a single antibody isotype–antigen combination, pipette
150 μL of dilution buffer in rows B-H of a U-bottom 96-well
disposable plate. For row A, 180 μL of dilution buffer is added
to each well. Add 20 μL of the test serum sample to a well in
Row A to create a starting 1:10 dilution (see Note 10). If
available, place positive control (e.g., serum from previously
tested animal) in well A11 and negative control in well A12
(e.g., serum from nonimmunized animal) and dilute as above.
Once all samples have been loaded, use a multichannel to
serially dilute 69.4 μL of diluted serum sample 7 times between
rows B-H by pipetting up and down 4–6 times (see Note 11).
5. If running three plates per sample, use a 0.5 mL deep-well assay
block plate and add 400 and 540 μL of dilution buffer to rows
B-H and A, respectively. 60 μL of the test serum would be
added to Row A to create a starting 1:10 dilution and the serial
dilutions would be performed by pipetting 185 μL of sample
down the rows.
Determination of Antigen-Specific IgG Antibody Titers
541