Radio Immuno assay

A 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.

1. RADIOIMMUNOASSAY Prasanna R Kovath Assistant Professor Department of Biotechnology St.Mary’s College Thrissur

2. Radioimmunoassay (RIA) is an  in vitro  assay that measures the presence of an antigen with very high sensitivity. When radioisotopes instead of enzymes are used as labels to be conjugated with antigens or antibodies, the technique of detection of the antigen-antibody complex is called radioimmunoassay (RIA).  A 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.

3. Principle of Radioimmunoassay It involves a combination of three principles. An immune reaction i.e. antigen, antibody binding. A competitive binding or competitive displacement reaction. (It gives specificity) Measurement of radio emission. (It gives sensitivity) RIA was first described in 1960 for the measurement of endogenous plasma insulin by  Solomon Berson and Rosalyn Yalow  of the Veterans Administration Hospital in New York. The classical RIA methods are based on the principle of competitive binding

4. An unlabeled antigen competes with a radiolabeled antigen for binding to an antibody with the appropriate specificity . Thus, when mixtures of radiolabeled and unlabeled antigen are incubated with the corresponding antibody, the amount of free (not bound to antibody) radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the mixture . Nanomolar and picomolar concentrations of hormones in biological fluids can be detected by RIA. It is the first immunoassay technique to analyze small amounts of analyte .

5. Requirements Specific antiserum to antigens for estimation Radioactively labeled antigens Some technique to separate the bound and unbound antigen-antibody complexes Instrumentation to measure and estimate radioactivity level. Radioisotopes used: Iodine isotope 125-I labels Although both carbon isotopes such as C14 and H3 have been used nowadays .

6. Radioimmunoassay Method In the basic method of Radioimmunoassay, we use the target antigen which is labeled radioactively and bound to its specific antibodies. We will require a limited and known antibody to be added in a specific amount in Radioimmunoassay. A sample is then added in order to initiate a reaction competitive in nature, of the labeled antigens from the preparation, and the unlabeled antigens from the sample, with the specific antibodies. This reaction to the antibodies will release a certain amount of labeled antigen. This amount is correlative to the ratio of labeled to unlabeled antigen. A binding curve thus obtained allows the amount of antigen in the patient's serum to be derived

7. Separation techniques A double antibody, charcoal, cellulose, chromatography or solid phase techniques are applied to separate bound and free radio-labeled antigen in radioimmunoassay. Most frequently used the technique for this is double antibody technique which is combined with polyethylene. The bound or free fraction is computed in a gamma counter. Collectively, a calibration or standard curve is generated with samples of known concentrations of the unlabeled standards. The presence and amount of antigen in an unknown sample can be calculated from this curve .

8. The  extremely high sensitivity  of RIA is its  major advantage The sensitivity for the radioimmunoassay experiment can be improved by decreasing the amount of radioactively-labeled antigen and/or antibody. The sensitivity of the radioimmunoassay can also be improved by the so-called disequilibrium incubation. For the same, the radioactively labeled antigen is added after initial incubation of antigen and antibody. https://youtu.be/JtbcqkiFf7w

9. Important Note The antibody must be specific for the antigen which is being Radioimmunoassay experimented upon. Make sure other antigens must And should not cross-react with the antibody used.

10. Applications Detection of drugs such as narcotics and morphine in hair samples. Estimation of mere amounts of hydromorphone and hydrocodone derivatives of morphine drug in blood sample. Detecting Flunisolide in blood plasma. Flunisolides is corticoid and designed for treatment of asthma, allergy and arthritis. Estimation of Ferritin in blood serum of patient. This Ferritin in high concentration is indicative of iron stores. Sometimes the body has the sufficient levels of iron yet the stored form of iron is not used for some metabolic condition. Therefore, RIA in such case is helpful to identify if there is truly iron deficiency in patient body or some metabolic issues. Thyroids testing by estimating the levels of thyroxin in blood. Detection of certain toxins in blood sample.