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Step 1 |
Adding Negative Control, Test Sample and Blank:
Add 50μl of Negative Control, 100μl of test sample and 100μl of 1 x Assay Buffer as Blank into their respective wells. Triplicate test is recommended for Negative Control and duplicate test is recommended for Blank and test samples. Note: Use a separate disposal pipette tip for each test sample, Negative Control and Blank to avoid cross-contamination. Mix by tapping the plate gently. |
| Step 2 | Incubation:
Cover the plate and incubate at room temperature for 1 hour. |
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Step 3 |
Washing:
Discard the content and tap the plate on a clean paper towel to remove residual solution in each well. Add 300 µl of 1×Wash buffer to each well and incubate for 1 minute. Discard the 1×Wash buffer and tap the plate on a clean paper towel to remove residual wash buffer. Repeat the wash step for a total 3 washes. |
| Step 4 | Adding HRP-conjugated Detection Solution:
Add 100 µl of 1×Detection Solution to each well. |
| Step 5 | Incubation:
Cover the plate and incubate at room temperature for 1 hour. |
| Step 6 | Washing:
Wash each well 4 times as described in step 3. |
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Step 7 |
Colouring:
Add 100 µl of Substrate solution to each well, incubate at room temperature for 15 minutes. Protect from light. |
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Step 8 |
Stopping Reaction:
Add 100 µl of Stop solution to each well, gently tap the plate frame for a few seconds to ensure thorough mixing. |
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Step 9 |
Measurement:
Measure absorbance of each well at 450 nm immediately. Note: read the absorbance within 10 minutes after stopping the reaction. |