1. Wash each well three times with 200 μL TBST. Add 100 μL of the biotinylated capture antibody to each well and incubate at room temperature for 2 hours.
2. Wash each well three times with 200 μL TBST. Make a serial dilution of the antigen, adding 100 µl per well. Incubate at room temperature for 1 hour.
3. Wash each well three times with 200 μL of TBST. Add 100 μL of primary antibody to each well and incubate plate at room temperature for 1 hour.
4. Wash each well three times with 200 μL of TBST. Add 100 μL of the enzyme-labeled secondary antibody to each well. Incubate plate for 30 minutes at room temperature.
5. Wash each well three times with 200 μL of TBST. Follow your manufacturer’s instructions for their specific detection system.