Biochemical Techniques and Instrumentation

3.1.6. Technique

• A known quantity of an antigen is made radioactive by labeling it with gamma-radioactive isotopes.
• The radiolabeled antigen is then mixed with a known quantity of specific antibody and they will chemically bind to one another.
• Then, the sample containing an unknown quantity of the same antigen or unlabeled antigen is then added.
• The unlabeled antigen also competes with the radiolabeled antigen for the antibody binding sites.
• If the concentration of unlabeled antigen is increased, it displaces the radiolabeled antigen and binds more to the antibody
• The ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen is then reduced.
• The bound antigens are then separated from the unbound antigens.
• The radioactivity of the free antigen remaining in the supernatant is then measured.
• A binding curve is then generated with the known standards and  the amount of antigen can be derived.

Figure