Optimal dilutions/concentrations should be determined by the end user.

Recommended Procedure:

1. Dilute the Capture Antibody to working concentration using Coating Buffer. Immediately coat the 96-well plate with diluted Capture Antibody (100 µl per well). Seal the plate and incubate at 4 °C overnight or at 37 °C for 2 hours
2. Aspirate the wells and wash with Wash Buffer (350 µl per well) and allow to soak for 1-2 min. Remove the liquid by inverting and tapping the plate on to absorbent paper.
3. Block the plate with Blocking Buffer (200 µl per well) at 37 °C for 1.5 hours.
4. Repeat the aspiration/wash process in Step 2.
5. Add 50 µl of standards or sample into the appropriate wells, followed by 50 µl of working biotin-conjugated Competitor. Cover with a plate sealer and incubate at 37 °C for 1 hour.
6. Repeat the aspiration/wash process in Step 2.
7. Add appropriately diluted Streptavidin HRP (100 µl per well). Cover the plate with a new plate sealer and incubate at 37 °C for 30 min.
8. Repeat the aspiration/wash process in Step 2, for a total of 5 times.
9. Add Substrate Solution (90 µl per well). Cover the plate with a new plate sealer and incubate at 37 °C for 10-20 min. Keep the plate in the dark and avoid exposure to light.
10. Add Stop Solution (50 µl per well). Tap the side of the plate to ensure thorough mixing.
11. Measure the absorbance immediately using a microplate reader set at 450 nm.