12. Flick off Blocking Buffer from plates and bang the plate onto
absorbent pads to remove air bubbles and excess moisture.
13. Add 100 μL/well of samples.
14. In a 24-well plate, prepare Initial dilution of Standard Serum.
Serially dilute serum in Blocking Buffer, Add 100 μL/well of
Standard dilutions and blank to plate.
15. Cover the plate with a plastic seal.
16. Incubate for 2 h at RT.
17. Gently flick contents of microplate into the sink and wash
plates six times with Washing buffer >250 μL/well each;
bang the plate onto absorbent pads to remove air bubbles
and excess moisture in between every two consecutive washes.
18. Dry all surfaces with a paper towel.
19. Dilute the biotinylated anti-mouse detection antibodies and
streptavidin-HRP in Blocking buffer (total volume required is
10 mL per plate). Prepare this just before washing plates.
20. Gently add 100 μL/well of the diluted detection antibody
using a multichannel pipette without forming air bubbles.
21. Cover the plate with a plastic seal.
22. Incubate for 1 h at RT.
23. Wash the plate six times.
24. Mix equal volume of Peroxidase Substrate and Peroxidase
Substrate Solution B just before use. 10 mL are required per
ELISA plate.
25. Add 100 μL/well of the TMB substrate solution using a multichannel pipette.
26. Incubate for 10 min at RT to allow color to develop.
27. Add 100 μL/well of stop solution using a multichannel pipette
to stop the reaction.
28. Gently tap the plate to stop the reaction uniformly.
29. Measure OD at 450 nm with a VersaMax ELISA microplate
reader (Molecular Devices, CA, USA) and analyzed using SoftMax Pro Software.
30. For determination of ELISA end-point titers, absorbance
cut-off values are established as the mean absorbance of eight
negative-control wells containing sera of naive mice plus 3 SD.
31. Absorbance values of test sera were considered positive if they
were equal to or greater than the absorbance cut-off and
end-point titers calculated as log 10 of the reciprocal of the last
dilution giving a positive absorbance value.
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Lei Li et al.
absorbent pads to remove air bubbles and excess moisture.
13. Add 100 μL/well of samples.
14. In a 24-well plate, prepare Initial dilution of Standard Serum.
Serially dilute serum in Blocking Buffer, Add 100 μL/well of
Standard dilutions and blank to plate.
15. Cover the plate with a plastic seal.
16. Incubate for 2 h at RT.
17. Gently flick contents of microplate into the sink and wash
plates six times with Washing buffer >250 μL/well each;
bang the plate onto absorbent pads to remove air bubbles
and excess moisture in between every two consecutive washes.
18. Dry all surfaces with a paper towel.
19. Dilute the biotinylated anti-mouse detection antibodies and
streptavidin-HRP in Blocking buffer (total volume required is
10 mL per plate). Prepare this just before washing plates.
20. Gently add 100 μL/well of the diluted detection antibody
using a multichannel pipette without forming air bubbles.
21. Cover the plate with a plastic seal.
22. Incubate for 1 h at RT.
23. Wash the plate six times.
24. Mix equal volume of Peroxidase Substrate and Peroxidase
Substrate Solution B just before use. 10 mL are required per
ELISA plate.
25. Add 100 μL/well of the TMB substrate solution using a multichannel pipette.
26. Incubate for 10 min at RT to allow color to develop.
27. Add 100 μL/well of stop solution using a multichannel pipette
to stop the reaction.
28. Gently tap the plate to stop the reaction uniformly.
29. Measure OD at 450 nm with a VersaMax ELISA microplate
reader (Molecular Devices, CA, USA) and analyzed using SoftMax Pro Software.
30. For determination of ELISA end-point titers, absorbance
cut-off values are established as the mean absorbance of eight
negative-control wells containing sera of naive mice plus 3 SD.
31. Absorbance values of test sera were considered positive if they
were equal to or greater than the absorbance cut-off and
end-point titers calculated as log 10 of the reciprocal of the last
dilution giving a positive absorbance value.
416
Lei Li et al.