1. Determine wells for diluted standard, blank and sample. Prepare 7 wells for standard, 1 well for blank.
Add 100μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate
wells. Cover with the Plate sealer. Incubate for 2 hours at 37oC.
2. Remove the liquid of each well, don’t wash.
3. Add 100μL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37oC after covering
it with the Plate sealer.
4. Aspirate the solution and wash with 350μL of 1× Wash Solution to each well using a squirt bottle,
multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1~2 minutes. Remove the remaining
liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. After the
last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against
absorbent paper.
5. Add 100μL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37oC after
covering it with the Plate sealer.
6. Repeat the aspiration/wash process for total 5 times as conducted in step 4.
7. Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at
37oC (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate
Solution.
8. Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the
liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure
thorough mixing.
9. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the
surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately.