1. Add 200 ul of PBS (or DW) to each well. Aspirate the wells
to remove liquid and wash the plate 3 times using 300 ul of
washing solution per well. After the last wash invert plate to
remove residual solution and blot on paper towel.
2. Add 100 ul of standard or sample to each well in duplicate.
Incubate at room temperature for at least 2 hours. (or 37°C
for 1 hour)
3. Aspirate the wells to remove liquid and wash the plate
4 times like as step 1.
4. Add 100 ul of the diluted detection antibody (0.25 ug/ml)
per well. Incubate at room temperature for 2 hours. (or 37°C
for 1 hour)
5. Aspirate and wash plate 4 times like as step 1.
6. Add 100 ul of the diluted color development enzyme (1/200
dilute) per well. Incubate 30 minutes at room temperature.
(or 37°C for 30 minutes)
7. Aspirate and wash plate 4 times like as step 1.
8. Apply 100ul of pink-ONE TMB to each well. After sufficient
color development (5-10minutes at room temperature or at
37°C), add 100 ul Stop Solution (2M H2SO4) to each well.
9. Using a microtiter plate reader, read the plate at 450 nm
wavelength.
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