Radioimmunoassay
  • Radioimmunoassay (RIA) is a 
   very sensitive in vitro assay 
   technique used to measure 
   concentrations of antigens (for 
   example, hormone levels in the 
   blood) by use of antibodies.
  • RIA technique is extremely 
   sensitive and extremely 
   specific, requiring specialized 
   equipment.
              History
   • The technique was introduced in 1960 by Berson 
    and Yalow as an assay for the concentration of 
    insulin in plasma.
   •  It represented the first time that hormone levels in 
    the blood could be detected by an in vitro assay.
   • The technique of radioimmunoassay has 
    revolutionized research and clinical practice in 
    many areas, e.g., 
    –Blood banking 
    –Diagnosis of allergies 
    –Endocrinology
                 Principle
    • The technique is based on the ability of an 
      unlabelled form of the substance to inhibit 
      competitively the binding of a 
      radioactively labelled substance by 
      specific antibodies.
                     I125 labeled 
                      antigen
   Ag in serum
    Ab for the Ag
    •   It is based on a principle of serial dilutions. One 
        starts with a combination of radioactively labelled 
        antigen (corresponding to the hormone to be 
        measured) and antibody to that hormone. 
    •   Then, a specific quantity of unlabeled, or cold 
        antigen is added to the mixture. The unlabeled 
        antigen competes with the radioactive antigen for 
        binding to the antibody and displaces a 
        proportional amount of it. The unbound antigen is 
        separated away (by centrifugation, for example) 
        and the amount of radioactivity remaining is 
        measured.
  • This process is continually repeated, 
   using progressively greater 
   concentrations of unlabeled antigen, and 
   a line graph demonstrating the 
   relationship between concentration of the 
   unlabeled antigen and the radioactivity 
   remaining is constructed. 
  • This process creates a standard binding 
   curve. The competitive binding process is 
                        then repeated using the 
   biological                               sample to be 
   tested and by comparison                   the 
   radioactivity  resulting with the                  
           standard binding curve, one can 
   deduce                        the concentration 
   of the hormone in the                   sample 
   of interest.