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Radioimmunoassay: Advanced Research Techniques in Basic Medical Sciences
Radioimmunoassay: Advanced Research Techniques in Basic Medical Sciences
Radioimmunoassay
Associate Professor Dr. zhan Eyigr Uludag University College of Medicine Department of Histology & Embryology
Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity
The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,
blood banking diagnosis of allergies endocrinology
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
A mixture is prepared of
radioactive antigen
Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125I or 131I are often used.
Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies.
At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured.
Gamma Counter
From these data, a standard binding curve, like the one shown in red, can be drawn.
The samples to be assayed (the unknowns) are run in parallel. After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve.
Radioimmunoassay is widely-used because of its great sensitivity. Using antibodies of high affinity, it is possible to detect a few picograms (1012 g) of antigen in the tube. The greater the specificity of the antiserum, the greater the specificity of the assay
The main drawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive antigen. Both 125I or 131I emit gamma radiation that requires special counting equipment; The body concentrates iodine atoms radioactive or not in the thyroid gland where they are incorporated in thyroxine (T4).
Despite these drawbacks, RIA has become a major tool in the clinical laboratory where it is used to assay plasma levels of:
most of our hormones; digitoxin or digoxin in patients receiving these drugs; certain abused drugs
for the presence of hepatitis B surface antigen (HBsAg) in donated blood; anti-DNA antibodies in systemic lupus erythematosus (SLE).
The molecule is detected by antibodies that have been made against it; that is, for which it is the antigen. Monoclonal antibodies are often used. The test requires:
the antibodies fixed to a solid surface, such as the inner surface of a test tube; a preparation of the same antibodies coupled to an enzyme. This is one (e.g., galactosidase) that produces a colored product from a colorless substrate.
Some examples:
detecting infections
sexually-transmitted agents like HIV, syphilis, and chlamydia hepatitis B and C Toxoplasma gondii
detecting allergens in food and house dust measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases like lupus erythematosus measuring toxins in contaminated food detecting illicit drugs, e.g.,
cocaine opiates -9-tetrahydrocannabinol, the active ingredient in marijuana