RIA is based on the principle of competitive binding. In this technique, competitive binding occurs between an unlabeled antigen and radiolabel antigen (radiolabel with 125I-$\gamma$ emitting, bind to a tyrosine of protein or 3H-β particle emitting) with the high-affinity antibody. Firstly antigen mix is labeled with known amount of antibody in that concentration, which is able to saturate the antigen binding site on antibody and through which we can calculate total count per minute (CPU) through beta or gamma counter, after that the amount of radiolabeled antigen is obtained which is required to saturate the known amount of antibody.