1. Prepare Standard and samples in Standard and Sample Diluent.
2. Add 100 uL of Standard or sample to appropriate wells.
3. Cover plate with Plate Sealer and incubate at room temperature (20-25 degrees C) for 1 hour.
4. Wash plate four times with 1X Wash Buffer.
5. Add 100 uL of Biotinylated Antibody Working Solution to each well.
6. Cover plate with Plate Sealer and incubate at room temperature for 1 hour.
7. Wash plate four times with 1X Wash Buffer.
8. Add 100 uL of Streptavidin-HRP Working Solution to each well.
9. Cover plate with Plate Sealer and incubate at room temperature for 30 minutes.
10. Wash plate four times with 1X Wash Buffer.
11. Add 100 uL of TMB Substrate to each well.
12. Develop the plate in the dark at room temperature for 30 minutes.
13. Stop reaction by adding 100 uL of Stop Solution to each well.
14. Measure absorbance on a plate reader at 450 nm.
