5. Enzyme immunoassay flat-bottom 96-well microplates (see
Note 4).
6. Multichannel pipettes.
7. 96-Well dilution plates (vinyl or polypropylene).
8. Microcentrifuge.
9. Centrifuge with plate rotor.
10. P200 and P1000 pipette tips.
11. 15 and 50 mL conical tubes.
12. Plastic wrap.
13. Microplate washer.
14. Microplate absorbance spectrophotometer.
3 Methods
3.1 Sample
Collection
1. Collect blood from immunized mouse in a serum separator
tube (see Note 5).
2. Incubate blood for 30 min at room temperature (RT) to allow
blood to clot.
3. Place tubes in a microcentrifuge and centrifuge blood at
13,000 Â g for 5 min at RT.
4. Collect the serum using a pipette and transfer to 1.2 mL cluster
tubes or 1.5 mL microcentrifuge tubes (see Note 6).
5. Serum can be frozen at <À15
C until ready for analysis.
3.2 Antigen Coating
1. Determine the number of plates required to test desired samples (each plate usually contains 12 test samples).
2. Prepare sufficient quantities of antigen solution (~10 mL per
plate) by diluting coating antigen (e.g., hemagglutinin) to a
final concentration of 1 μg/mL in PBS (see Note 7).
3. Dispense 100 μL of antigen solution to each well using multichannel pipette.
4. Place plates in stacks of up to ten plates and cover stack with
plastic wrap. Incubate overnight at RT.
5. In preparation for Subheading 3.4, thaw serum samples at 4
C
overnight. Alternatively, samples can be thawed at room temperature during the blocking step (Subheading 3.3, step 3).
3.3 Blocking
of Plates
1. On the following day (16–20 h postincubation), wash the
plates 5Â with 200 μL per well of 1Â Wash Buffer solution
using the automated plate washer. Washes can also be done by
hand using a multichannel pipette (see Note 8).
540
Bassel Akache et al.
Note 4).
6. Multichannel pipettes.
7. 96-Well dilution plates (vinyl or polypropylene).
8. Microcentrifuge.
9. Centrifuge with plate rotor.
10. P200 and P1000 pipette tips.
11. 15 and 50 mL conical tubes.
12. Plastic wrap.
13. Microplate washer.
14. Microplate absorbance spectrophotometer.
3 Methods
3.1 Sample
Collection
1. Collect blood from immunized mouse in a serum separator
tube (see Note 5).
2. Incubate blood for 30 min at room temperature (RT) to allow
blood to clot.
3. Place tubes in a microcentrifuge and centrifuge blood at
13,000 Â g for 5 min at RT.
4. Collect the serum using a pipette and transfer to 1.2 mL cluster
tubes or 1.5 mL microcentrifuge tubes (see Note 6).
5. Serum can be frozen at <À15
C until ready for analysis.
3.2 Antigen Coating
1. Determine the number of plates required to test desired samples (each plate usually contains 12 test samples).
2. Prepare sufficient quantities of antigen solution (~10 mL per
plate) by diluting coating antigen (e.g., hemagglutinin) to a
final concentration of 1 μg/mL in PBS (see Note 7).
3. Dispense 100 μL of antigen solution to each well using multichannel pipette.
4. Place plates in stacks of up to ten plates and cover stack with
plastic wrap. Incubate overnight at RT.
5. In preparation for Subheading 3.4, thaw serum samples at 4
C
overnight. Alternatively, samples can be thawed at room temperature during the blocking step (Subheading 3.3, step 3).
3.3 Blocking
of Plates
1. On the following day (16–20 h postincubation), wash the
plates 5Â with 200 μL per well of 1Â Wash Buffer solution
using the automated plate washer. Washes can also be done by
hand using a multichannel pipette (see Note 8).
540
Bassel Akache et al.