• Pre-coated 96-well microplate
• Wash Buffer
• Assay Diluent
• Ligand Protein
• Detection Antibody
• HRP-Streptavidin Solution
• TMB One-Step Substrate Reagent
• Stop Solution

• Microplate reader capable of measuring absorbance at 450 nm
• Shaker
• Pipettes capable of accurately delivering 2 µL to 1 mL volumes
• Pipettes capable of delivering 1-25 mL volumes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Distilled or deionized water
• Tubes to prepare sample dilutions

1. Prepare all reagents and test reagent samples as instructed.
2. Add 100 µL test reagent samples to each well. Incubate 2.5 hours at room temperature or overnight at 4°C.
3. Add 100 µL the prepared Detection Antibody solution to each well. Incubate 1 hour.
4. Add 100 µL the prepared HRP-Streptavidin solution to each well. Incubate 30 minutes.
5. Add 100 µL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes.
6. Add 50 µL Stop Solution directly to each well. Read at 450 nm immediately.

The kit may be stored at 4°C up to 1 month from the date of shipment. Shelf life can be extended up to 6 months by storing protein and antibodies, at -20°C immediately upon delivery. All other kit components should be stored at 4°C.