Radioimmunoassay

a detailed presentation on RIA technique.

1. RADIOIMMUNOASSAY BY: FARHA BANU, MSC MICROBIOLOGY

2. 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

3. ANTIGENS An antigen is a substance with the ability to induce an immunological response. They typically enter the body from an infection. They are recognized at their epitopes by B cells or by the T cell receptor on T cells.

4. ANTIBODIES Antibodies are produced by the B-lymphocytes. They are glycoproteins belonging to the “immunoglobulin supergene family” that are produced in response to a foreign substance in the body. • Antibodies have a generally common structure, but have regions that vary among them to accommodate the specific antigens.

5. LABELS IN RIA Immunoassays require the use of labeled materials in order to measure the amount of antigen or antibody present. A label is a molecule that will react as part of the assay, and in doing so produce a signal that can be measured in the solution. Examples of a label include a radioactive compound, or an enzyme that causes a change of color in a solution or its fluorescence .

6. INTRODUCTON Radio immune assay (RIA): As the name indicates, it is an  immunological assay  to analyze any antigen or anti-body in the patient’s serum to diagnose the disease. This is one of the most sensitive & specific methods of immune assays available. It involves competitive binding of radio-labeled antigen and unlabeled antigen to a high-affinity antibody. The sensitivity range is 0.0006–0.006 µg antibody/ml.

7. PRINCIPLE It involves three principles which make it most specific & sensitive than other immune assays. 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)

8. PRINCIPLE IMMUNE REACTION:  When a foreign biological substance enters into body blood stream through non oral route, body recognizes the specific chemistry on surface of foreign substance as antigen and produces specific antibodies against the antigen so as nullify the effects and keep the body safe. The antibodies are produced by body immune system so, it is an immune reaction.

9. PRINCIPLE 2. A competitive binding or competitive displacement reaction: This is a phenomenon wherein when there are two antigens which can bind to same antibody, the antigen with more concentration binds extensively with the limited antibody displacing other. So here in the experiment, radiolabelled antigen is allowed to bind to high affinity antibody. Then when patient serum is added unlabelled antigens in it start binding to the antibody displacing the labeled antigen.

10. PRINCIPLE 3.Measurement of radio emission:  Once the incubation is over, then washings are done to remove any unbound antigens. Then radio emission of the antigen antibody complex is taken, the gamma rays from radio labeled antigen are measured.

11. APPROCH FOR RIA The first step to set up an RIA is to determine the amount of antibody needed to bind 50–70% of a fixed quantity of radioactive antigen (Ag*) in the assay mixture. This ratio of antibody to Ag* is chosen to ensure that the number of epitopes presented by the labeled antigen always exceeds the total number of antibody binding sites. Consequently, unlabeled antigen (from patient serum) added to the sample mixture will compete with radiolabeled antigen to bind to the limited number of antibody.

12. Even a small amount of unlabeled antigen added to the assay mixture of labeled antigen and antibody will cause a decrease in the amount of radioactive antigen bound, and this decrease will be proportional to the amount of unlabeled antigen added. To determine the amount of labeled bound antigen, the Ag- Ab complex is precipitated to separate it from free antigen (antigen not bound to Ab ), and the radioactivity in the precipitate is measured. A standard curve can be generated using unlabeled antigen samples of known concentration (in place of the test sample), and from this plot the amount of antigen in the test mixture may be precisely determined.

13. PROCEDURE

14. PROCEDURE 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.

15. PROCEDURE 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.

16. PROCEDURE The antigen is generally labeled with a gamma-emitting isotope such as I 125 , but beta-emitting isotopes such as tritium (3H) are also routinely used as labels. The radiolabeled antigen is part of the assay mixture; the test sample may be a complex mixture, such as serum or other body fluids, that contains the unlabeled antigen.

17. ADVANTAGES & DISADVANTAGES ADVANTAGES Highly specific: Immune reactions are specific. High sensitivity:Immune reactions are sensitive. DISADVANTAGES Radiation hazards. Requires specially trained persons. Lab requires special license to handle radioactive material. Storage and disposal of radioactive material.

18. APPLICATIONS Detection of narcotic drugs. Measurement of growth hormone levels, immunoglobulins . Allergy diagnosis. Detect drug abuse or drug poisoning. Early cancer detection and diagnosis. Blood bank screening for the hepatitis virus.

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