RADIO IMMUNO ASSAY

A technique for determining antibody levels by introducing an antigen labelled with a radioisotope and measuring the subsequent radioactivity of the antibody component.

1. RADIO IMMUNO ASSAY

2. R . I . A (RADIO IMMUNO ASSAY) a technique for determining antibody levels by introducing an antigen labelled with a radioisotope and measuring the subsequent radioactivity of the antibody component .

3. INTRODUCTION RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies. • The technique was first developed in 1960 by two endocrinologists, S. A. Berson and Rosalyn Yalow , to determine levels of insulin–anti-insulin complexes in diabetics. • In 1977, some years after Berson’s death, the significance of the technique was acknowledged by the award of a Nobel Prize to Yalow.

4. PRINCIPLE • The principle of RIA involves competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody. • The labeled antigen is mixed with antibody at a concentration that saturates the antigen-binding sites of the antibody. • Then test samples of unlabeled antigen of unknown concentration are added in progressively larger amounts .

5. • The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody. • As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites . • The decrease in the amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample.

6. The antigen is generally labeled with a gamma emitting isotope such as 125 I , buT beta-emitting isotopes such as tritium ( 3 H ) and 14 C are also routinely used as labels.

7. REAGENTS USED IN RID A tracer i.e. a labelled ligand . 2. A binder (Antibody) which is the specific antiserum. 3. A separation system to separate the ‘bound’ and ‘free’ phases. 4. A standard (in highly pure form) . 5. A free human antiserum.

8. PROCEDURE

9. STEPS

10.  A known quantity of an antigen is made radioactive . This radiolabeled antigen is then mixed with a known amount of antibody for that antigen , and as a result, the two chemically bind to one another. 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 for antibody binding sites . As the concentration of "cold" antigen is increase, more of it binds to the antibody . 

11. 6. By displacing the radio labelled variant , reduces the ratio of antibody-bound radio labelled antigen to free radio labelled antigen. 7. The bound antigens are then separated from the unbound ones . 8. the radioactivity of the free antigen remaining in the supernatant is measured. HERE separating bound from unbound antigen is crucial .

12. ADVANTAGES Radioimmunoassay is widely used because of its great sensitivity . • Using antibodies of high affinity, it is possible to detect a few picograms of hormone to the tube. • The greater the specificity of the antiserum, the greater the specificity of the assay .

13. DISADVANTAGES • Prolonged reaction time (in days) as a consequence highly diluted reagent is used. •Radioactive Iodine used in is not a cheap reagent . •Possible health hazards due to handling of radioisotopes. •All the reagents must be added precisely. • Limited assay range. • Difficulty of automation . • Lengthy counting time.

14. USES OF RIA   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 , and Research with brain chemicals called neurotransmitters.

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