1.Calculate the desired number of microwells. Remove the remaining microwells and place them with desiccant into the
resealable plastic bag, seal and store at 2-8°C for later use.
2.Add Calibrators and Samples:
2. 1 Blank well: Do not add reagents.
2. 2 Calibrators wells: Add 100 µL of Calibrators into the designated control wells.
2.3 Test wells: Add 100 uL Sample Diluent to the test wells, then add 10 uL sample in the corresponding well.
2.4 Gently shake the plate for 20 seconds, and then cover the plate with the sealer.
3. Incubate:Incubate the plate at 37°C for 30 minutes.
4. Wash Step (Can be performed manually or with automated washing):
Manual washing: Carefully remove the incubation mixture by disposing of the solution into a waste container. Fill each
well with 350 µL diluted wash buffer and shake gently for 20-30 seconds. Discard the wash solution completely. Repeat 4
more times. After completing the last wash step, tap the plate on absorbent paper to remove residual liquid.
Automated washing: Automatic plate washer must be calibrated to ensure efficient washing. Fill each well with 350 µL
diluted wash buffer and soak for 20-30 seconds. Aspirate all wells completely. Repeat 4 more times.
5. Conjugate: Add 100 μL conjugate into each well except the Blank Well.
6. Incubate: Cover the wells and incubate at 37°C for 30 minutes.
7. Wash Step: Wash the plate 5 times as described in step 4.
8. Substrate: Add 50 µL of Substrate A and 50 µL of Substrate B into each well, including the Blank Well. Gently shake the
microwellsfor 20 seconds. Incubate at 37°C in the dark for 15 minutes.
9. Stop Solution: Add 50 μL Stop Solution to each well.A yellow color should develop in wells containing Positive specimens.
10. Read Results: Read at 450/630-700 nm within 30 minutes.
1. Note: Microplate can also be read at 450 nm, but it is strongly recommended to read it at 450/630-700 nm for
better results.
Chronometry: Read the sample's optical density (OD) at 450nm with a micro plate reader.
The Mean negative control OD value ≤ 0.1 and Mean positive control OD value ≥ 0.8, the test is valid,
otherwise the test is invalid.
Cut-Off value (C.O.) = The Mean OD value of negative control x 3 (Calculated by 0.10 when the Mean
negative control OD value is < 0.10, calculated by actual value when the Mean negative control OD
Positive Results: Sample OD value ≥ C.O.
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