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Radioimmuno assay

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Radioimmuno assay

  1. 1. Radioimmuno Assay Presented by : Mamona Waheed Presented to : Sir Alamgeer
  2. 2. 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.
  3. 3. • RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains the least expensive method to perform such tests
  4. 4. • 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.
  5. 5. • The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.G., • Blood banking • Diagnosis of allergies • Endocrinology
  6. 6. Radioimmunoassay: pros and cons • PRO: versatility : using the same principle, almost any biomolecule can be assayed • Fast (usually 2 days or less) • Sensitive (comparable to the most sensitive bioassays, that is < ng/ml) • Large capacity : thousands of samples/day specific (antibody- dependent)
  7. 7. Con: • Use of radioactivity: hazardous • Expensive equipment (gamma or beta • Counter)
  8. 8. •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.
  9. 9. Method • To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine (hot). • This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one another
  10. 10. • Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. • This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites.
  11. 11. • As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen.
  12. 12. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter. Using known standards, a binding curve can then be generated which allows the amount of antigen in the patient's serum to be derived.
  13. 13. The principle of RIA • The amount of Ab per tube is kept constant, the amount of antigen added (known or unknown) is the variable parameter. • The added antigen will be distributed between a bound (B) and a free (F) fraction. This distribution is governed by the association constant (KA) of the Ab: Ab + Ag 􀁺 AgAb and K = [AbAg] /[Ab][Ag]
  14. 14. • Conclusion: If total Ab input is kept constant, the value of B/F is a measure for the total Ag input
  15. 15. • To measure this distribution B-F , • A small but constant amount g p of labeled antigen ("tracer") is added to the reaction. • Eventually, there will be a competition reaction between this small but constant amount of "tracer" and the "cold" antigen for a limited amount of antibody.
  16. 16. Requirements for the development of an RIA 1. Pure antigen : for - standards (μg), - Tracer production (tens of μg) - Ab production (hundreds of μg)
  17. 17. 2. Tracer : self-made or commercial. 3. Specific, high-affinity antibody : self- made or commercial. 4. A method to separate bound and free antigen. 5. (Optional) : A system to extract the antigen from the sample.
  18. 18. Separating Bound from Free Antigen • Precipitate the antigen-antibody complexes by adding a "second" antibody directed against the first. For example, if a rabbit igg is used to bind the antigen, the complex can be precipitated by adding an antirabbit- igg antiserum (e.G., Raised by immunizing a goat with rabbit igg). This is the method shown in the diagram above.
  19. 19. • 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.
  20. 20. Radioimmunoassay is widely-used because of its great sensitivity.
  21. 21. • The greater the specificity of the antiserum, the greater the specificity of the assay.
  22. 22. Drawback • Expense and hazards of preparing and handling the radioactive antigen.
  23. 23. • 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).
  24. 24. RIA as a major clinical tool 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).
  25. 25. • Narcotics (drug) detection • Blood bank screening for the hepatitis (a highly contagious condition) virus • Early cancer detection • Measurement of growth hormone levels tracking of the leukemia virus • Diagnosis and treatment of peptic ulcers research with brain chemicals called neurotransmitters.

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