Remove the coating solution and wash the plate three times by filling the wells with 100 μL PBS-0.05 % Tween20. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.
Block the remaining protein-binding sites in the coated wells by adding 100 μL blocking buffer, 3 % skim milk in PBS per well. Incubate for 1 hour at RT with gentle shaking.
Wash the plate three times with 100 μL PBS-0.05 % Tween 20.
Add 50 μL of diluted antibody to each well. Incubate the plate at 37 °C for an hour with gentle shaking.
Wash the plate six times with 100 μL PBS-0.05 %Tween 20.
Add 50 μL of conjugated secondary antibody, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use. Incubate at 37 °C for an hour.
Wash the plate six times with 100 μL PBS-0.05 %Tween20.
Prepare the substrate solution by mixing acetic acid, TMB and 0.03 % H2O2 with the volume ratio of 4:1:5.
Dispense 50 μL of the substrate solution per well with a multichannel pipe. Incubate the plate at 37 °C in dark for 15-30 mins.
After sufficient color development, add 100 μL of stop solution to the wells (if necessary).
Read the absorbance (optical density at 450nm) of each well with a plate reader.