1. Antigen: Target proteins adhere to well bottom.
2. Wash: Removes unbound proteins to prevent false positives.
3. Primary Antibody: Specific to target antigen.
4. Secondary Antibody: Binds to primary. Has a linked enzyme.
5. Substrate (TMB): Enzyme converts colorless substrate to blue product.
Key Idea
If the patient is negative (no antigen), the primary antibody has nothing to grab. It will be washed away. Consequently, the secondary antibody has nothing to grab, and washes away. The final substrate will remain colorless.