A diagram showing the basic steps of an assay. 1 Antigen (blue) is bound to the surface of a plate. 2. Unbound antigen is washed away. 3. The sample containing the antibody to be tested (yellow) is added to the plate and binds to the antigen. 4. Unbound antibody is washed away. 5. A ligand is added that binds to the antibody – the ligand includes an enzyme. 6. Any unbound ligand is washed away. 7. A colourless substrate for the enzyme (chromogen) is added to the plate and left to develop. 8. The enzyme converts the chromogen into a coloured end-product that can be measured. The limiting factor in the ‘sandwich’ that is built up in the ELISA, is the amount of antibody; so the amount of coloured end-product is proportional to the amount of test antibody in step 3.