14. Add 100 μl of TMB substrate to each well, using the same pipetting order as appli ed in
step 7. Do not touch the side or bottom of the wells.
15. Cover the tray and incubate the tray for 30 minutes at room temperature. It is advised to
control the reaction on the plate regularly. In case of strong development the TMB
reaction can be stopped sooner. Avoid exposing the microwell strips to direct sunlight.
Covering the plate with aluminium foil is recommended.
16. Stop the reaction by adding 100 μl of stop solution with the same sequence and timing
as used in step 14. Mix solutions in the wells thoroughly by gently swirling the plate.
Gently tap the tray to eliminate any air bubbles trapped in the wells.
17. Read the plate within 30 minutes after addition of stop solution at 450 nm using a plate
reader, following the instructions provided by the instrument’s manufacturer.
*)
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