Radio Immune Eassay

Radio Immune Eassay

1. RadioImmuneAssay (RIA) By: Sondos Hassouneh Areej Abu hanieh Shireen Rawajbeh 1

2. IMMUNOASSAYS: 2

3. Immuno ” refers to an immune response that causes the body to generate antibodies Assay ” refers to a test. . Immunoassays are chemical tests used to detect or quantify a specific substance, the analyte , in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. High sensitivity is achieved by using an indicator system (e.g., enzyme label) that results in amplification of the measured product. Immunoassays may be qualitative (positive or negative) or quantitative (amount measured ).   Definition : 3

4. The purpose of an immunoassay is to measure (or, in a qualitative assay, to detect) an analyte . Immunoassay is the method of choice for measuring analytes normally present at very low concentrations that cannot be determined accurately by other less expensive tests Common uses include measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury 4

5. ANTIBODIES and ANTIGENS: An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. An antigen is a substance with the ability to induce an immunological response. 5

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7. LABELS IN IMMUNOASSAYS 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 substance that produce light . 7

8. Labels may be applied to either the antibody . . or the antigen . 8

9. Categories of Immunoassay Tests Competitive Noncompetitive Homogeneous Heterogeneous 9

10. 10 Competitive Assays: Unlabeled analyte (antigen) in the test sample is measured by its ability to compete with the labeled antigen in the immunoassay. In a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present.

11. Noncompetitive ( sandwhich ) immunoassays generally provide the highest level of assay sensitivity and specificity. This format is referred to as a “sandwich” assay because the analyte is bound (sandwiched) between two highly specific antibody reagents. The reaction mixture typically includes an excess of labeled antibody, so that all drug/metabolite is bound. The amount of antibody-antigen complex is then measured to determine the amount of drug present in the sample. The measurement of labeled analyte , usually antibody, is directly proportional to the amount of antigen present in the sample. Noncompetitive Assays 11

12. Heterogeneous and Homogeneous immunoassays Methods HETEROGENEOUS IMMUNOASSAYS : Called separation assay Require multiple steps Unbound analyte is separated or washed away, and the remaining labelled , bound analyte is measured. Those that do not require separation are referred to as HOMOGENEOUS IMMUNOASSAYS. Homogeneous methods have generally been applied to the measurement of small analytes such as Ab used and therapeutic drugs 12

13. There are several different methods used in immunoassay tests: Radioimmunoassay (RIA) Enzyme Immunoassay (ELISA) Fluorescence Polarization Immunoassay (FPIA) 13

14. it begin with the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantify it using radioactivity . The technique begin in 1960 by berson and yalow to test the blood Insulin 14

15. The technique of radioimmunoassay used in many research and clinical practice in many areas : Endocrinology 15

16. Principle: Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand Ag + Ag* + Ab  AgAb + Ag* Ab + Ag + Ab * Unbound Ag* and Ag washed out Radioactivity of bound residue measured Ligand conc. is inversely related to radioactivity [Ag : ligand to be measured ; Ag* radiolabelled ligand ] Principle of Radioimmunoassay 16

17. A mixture is prepared of radioactive antigen antibodies against that antigen. Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies. 17

18. + + P P* P* Q Radioactive tag Analyte Binding agent Free Bound Q P Q 18

19. 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 The radioactivity of each is measured. 19

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22. Preparation & characterisation of the Antigen [Ligand to be analysed] Radiolabelling of the Antigen Preparation of the Specific Antibody Development of Assay System Requirements of RIA 22

23. Antigens prepared by.. Synthesis of the molecule Isolation from natural sources Radiolabelling [Tagging procedure] 3 H , 14 C, 125 I are used as radioactive tags Antigens are tagged to 3 H , 14 C, 125 I Tagging should NOT affect Antigenic specificity & Antigenic activity ! Preparation and radiolabeling of the antigen 23

24. Antigen injected intradermally into rabbits or guinea pigs  antibody production Antibodies recovered from the serum. Preparation of the specific antibody 24

25. Crucial step is separation of unbound antigens Antibodies bind to microtitre well surface [Solid phase RIA] Antigens bound to the fixed antibodies remain stuck to the inner surface Decanting & washing the well removes unbound antigens Other techniques of separation: Centrifugation, Precipitation and Electrophoresis Development of the assay system 25

26. Video for RIA https:// www.youtube.com/watch?v=HOHczdglqHI https://www.youtube.com/watch?v=q4dY52v-IsY 26

27. Radioimmune procedure 27

28. From these data, a standard binding curve, like the e one shown in red, can be drawn. The samples to be assayed (the unknowns) are run in parallel. Calibration Curve 28

29. General uses In Endocrinology Insulin, HCG, Vasopressin Detects Endocrine Disorders Physiology of Endocrine Function In Pharmacology Morphine Detect Drug Abuse or Drug Poisoning Study Drug Kinetics 29

30. Clinical Immunology Antibodies for Inhalant Allergens Allergy Diagnosis Oncology Carcinoembryonic Antigen Early Cancer Detection and Diagnosis 30

31. tracking of leukemia virus diagnosis and treatment of peptic ulcers research with Neurotransmitters 31

32. Screening donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) Hepatitis C (presence of antibodies) Hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) APPLICATIONS 32

33. Negative : Positive: 33

34. LH (Luteinizing hormone , determining the time of ovulation) TSH, T3 and T4 (for thyroid function) Hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes. Detecting infections sexually-transmitted agents like HIV, syphilis, and chlamydia hepatitis B and C Toxoplasma gondii 34

35. 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. 35

36. RAST rating IgE level (KU/L) COMMENT < 0.35 Absent or undetectable allergen specific IgE 1 0.35 - 0.69 Low level of allergen specific IgE 2 0.70 - 3.49 Moderate level of allergen specific IgE 3 3.50 - 17.49 High level of allergen specific IgE 4 17.50 - 49.99 Very high level of allergen specific IgE 5 50.0 - 100.00 Very high level of allergen specific IgE 6 > 100.00 Extremely high level of allergen specific IgE 36

37. Measuring "rheumatoid factors" and other auto antibodies in autoimmune diseases like lupus erythematosus . Measuring toxins in contaminated food Detecting illicit drugs, e.g., cocaine opiates Δ-9-tetrahydrocannabinol, the active ingredient in marijuana 37

38. Detection of drug: 38

39. ADVANTAGES Radioimmunoassay is widely-used because of its great sensitivity. Using antibodies of high affinity, it is possible to detect a few pictograms (10 −12 g) of antigen in the tube. The greater the specificity of the antiserum, the greater the specificity of the assay 39

40. RIA has become a major tool in the clinical laboratory where it is used to assay . plasma levels of: most of our hormones; digitoxin or digoxin in patients receiving these drugs; certain abused drugs. Presence of hepatitis B surface antigen ( HBsAg ) in donated blood. Anti-DNA antibodies in systemic lupus erythematosus (SLE). 40

41. DRAWBACKS The main drawbacks to radioimmunoassay are the expense and hazards i f preparing and handling the radioactive antigen. Both 125 I or 131 I 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). 41

42. Labs require special license to handle radioactive material Requires special arrangements for Requisition storage of radioactive material radioactive waste disposal. 42

43. http://www.discoveriesinmedicine.com/Ni-Ra/Radioimmunoassay-RIA.html http://www.encyclopedia.com/medicine/divisions-diagnostics-and-procedures/medicine/radioimmunoassay Textbook of microbiology by Prescott, Harley. Practical analytical chemistry by BECKEET. http://www.millipore.com/immunodetection/id3/radioimmunoassay. References 43

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