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Radioimmunoassay

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. Properties.

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Radioimmunoassay

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  1. 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.

  2. Properties • RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains the least expensive method to perform such tests. • The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.

  3. 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 invitro assay. Dr. Rosalyn Yalow became the first female to win a Nobel Prize with her work on the radioimmunoassay

  4. 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. • Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ag*

  5. LABELS IN IMMUNOASSAYS • Used to detect reaction which has occurred Most common are: • Radioactive • Enzymes • Fluorescent • Chemiluminescent

  6. Radioimmunoassay (RIAs) utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.

  7. The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g., • Blood banking • Diagnosis of allergies • Endocrinology

  8. ADVANTAGES • Radioimmunoassay is widely-used because of its great sensitivity. • Using antibodies of high affinity, it is possible to detect a few picograms(10−12 g) of antigen in the tube. • The greater the specificity of the antiserum, the greater the specificity of the assay

  9. RIA has become a major tool in the clinical laboratory where it is used to assay . • Plasma levels of: • Most of our hormones; • Drugs like Digitoxin or digoxin; • Certain abused drugs like cocaine , opiates • Infectious Diseases like HBs Ag, HIV, TORCH. • Autoimmune Disease like Anti-DNA antibodies in systemic lupus erythematosus (SLE). • Allergy (RAST) • Measuring toxins in contaminated food

  10. 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. • antibodies against that antigen. • Knownamounts of unlabeled ("cold") antigenare added to samples of the mixture. These compete for the binding sites of the antibodies.

  11. 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 • determine the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve.

  12. The antigen-specific antibodies can be coupled to the inner walls of a test tube • After incubation, • The contents ("free") are removed • The tube is washed ("bound"), • The radioactive of both is measured. • The antigen-specific antibodies can be coupled to particles, like sephadex. Centrifugation of the reaction mixture separates the bound counts (in the pellet) from the free counts in the supernatant fluid.

  13. Count gamma emission • Counts per minute (CPM) for each tube • A sample containing a higher concentration of the unknown antigen will have a lower CPM • Commonly used radio isotopes… • I125(gamma emitting isotopes) • C14 and H3(Beta emitting isotopes)

  14. Gamma Counter

  15. From these data, a standard binding curve, like thee one shown in red, can be drawn. The samples to be assayed (the unknowns) are run in parallel. CALIBRATION CURVE

  16. Plot of Bound versus Total Drug Concentration Logit versus Log Total C Plot

  17. Detecting allergens in food and house dust . RAST: The radioallergosorbent test to detect specific IgE antibodies to suspected or known allergens . IgE is the antibody associated with type I allergic response : Pollen (is a fine to coarse powder containing the microgamatophytes of seeds) The amount of radioactivity is proportional to the serum IgE for the allergen.

  18. Disadvantages: • 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).

  19. Thankyou

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