Separation System:

It is the most important step in RIA procedure especially because of low concentrations involved. As already pointed out earlier, antigen is comparatively a smaller molecule whereas antibody is a macromolecule. Hence, Ag- Ab will differ in size considerably from the free antigen. A variety of physicochemical methods are employed for the separation of Ag-Ab complex from antigen. Most widely used method is precipitation of Ag-Ab by using polyethylene glycol. Another method is the solid phase separation technique. Some experimental factors such as pH, ionic strength, and high temperature, composition of the incubating medium buffer, heparin, urea, and high bilirubin concentrations are of special importance. The separated complex is assayed for its activity using a scintillation counter. Finally concentration of antigen is calculated.