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  2. 2. IMMUNOASSAYS “Immuno” refers to an immune response that causes the body to generate antibodies. “Assay” refers to a test.. An Immunoassay is a test that uses immunocomplexing when antibodies and antigens are brought together. 2
  3. 3. Categories of Immunoassay Tests Competitive Noncompetitive Homogeneous Heterogeneous 3
  4. 4. Labels may be applied to eitherthe antibody ..orthe antigen. 4
  5. 5. 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. 5
  6. 6. • Noncompetitive assay formats give the highest level of sensitivity and specificity. • They are normally used to measure critical analytes such as cardiac and hepatitis markers. Noncompetitive Assays • The measurement of the labeled analyte (Ab) α amount of Ag present in the sample 6
  7. 7. • There are two versions of the Non competitive format: • One step format Two step format 7
  8. 8. Heterogeneous and Homogeneous immunoassays Methods • Immunoassays that require separation of the bound Ab-Ag* complex are referred to as being HETEROGENEOUS IMMUNOASSAYS. • 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. 8
  9. 9. Radioimmunoassay (RIA) Enzyme Immunoassay (ELISA) Fluorescence Polarization Immunoassay (FPIA) 9
  10. 10. Radioimmunoassay (RIA) 10
  11. 11. •The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. •Here radioactive materials are not administered to the individual but are used as reagents. Radioimmunoassay (RIA) • Involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantify it using radioactivity 11
  12. 12. The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g., blood banking diagnosis of allergies endocrinology 12
  13. 13. Principle of RadioimmunoassayPrinciple of Radioimmunoassay 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] 13
  14. 14. + +P P* P*Q Radioactive tag Analyte Binding agent Free Bound Q PQ 14
  15. 15. 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. 15
  16. 16.  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. 16
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  19. 19. Requirements for RIARequirements for RIA 1. Preparation & characterisation of the Antigen [Ligand to be analysed] 2. Radiolabelling of the Antigen 3. Preparation of the Specific Antibody 4. Development of Assay System 19
  20. 20. Preparation & Radiolabelling of the AgPreparation & Radiolabelling of the Ag 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 ! 20
  21. 21. Preparation of the Specific AntibodyPreparation of the Specific Antibody Antigen injected intradermally into rabbits or guinea pigs  antibody production Antibodies recovered from the serum 21
  22. 22. Development of the Assay SystemDevelopment of the Assay System  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 22
  23. 23. Assay ProcedureAssay Procedure Known amounts of the test sample + labelled antigen into the microtitre wells Decant & wash contents of the well Measure radioactivity remaining in the Microtitre wells [GM counter , Scintillation counter etc] 23 incubate Intensity of radioactivity α 1/ conc. of Ag in test sample
  24. 24. Radioimmunoassay Procedure 24
  25. 25. Advantages & Disadvantages ofAdvantages & Disadvantages of RIARIA Advantages ◦ Highly specific: Immune reactions are specific ◦ High sensitivity : Immune reactions are sensitive ◦ Possible to detect picograms of Ag ◦ Sepharose beads used in RIA are reuseable 25
  26. 26. Disadvantages Radiation hazards: Uses radio labelled reagents Requires specially trained persons Labs require special license to handle radioactive material Requires special arrangements for Requisition storage of radioactive material radioactive waste disposal. 26
  27. 27. •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). 27
  28. 28. RIA EIA Sensitivity Nanomolar to picomolar Millimolar Cost More Less Time duration More Less Ease of handling Tedious Easy Radiation hazards More No Disposal Care has to be taken Easy Equipment Complex Less Mechanism Based on the measurement of Based on measurement of28
  29. 29. Applications ofApplications of RIARIA  Analysis of hormones, vitamins,metabolites, diagnostic markers ◦ Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids, Therapeutic drug monitoring: ◦ Barbiturates, morphine, digoxin,  Diagnostic procedures for detecting infection ◦ HIV, Hepatitis A, B etc 29
  30. 30. • 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; 30
  31. 31.  In Endocrinology Insulin, HCG, Vasopressin Detects Endocrine Disorders Physiology of Endocrine Function  In Pharmacology Morphine Detect Drug Abuse or Drug Poisoning Study Drug Kinetics 31
  32. 32.  Epidemiology Hepatitis B  Clinical Immunology Antibodies for Inhalant Allergens Allergy Diagnosis  Oncology Carcinoembryonic Antigen Early Cancer Detection and Diagnosis 32
  33. 33.  Narcotic drug detection  tracking of leukemia virus  diagnosis and treatment of peptic ulcers  research with Neurotransmitters 33
  34. 34. References •Yalow R, Berson S. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest 1960; 39: 1157-1175. •Abraham G. Radioimmunoassay of steroids in biological fluids. J. Steroid Biochemistry 1975; 6: 261-270. • Associate Professor Dr. Özhan Eyigör, Uludag University College of Medicine,Department of Histology & Embryology Radioimmunoassay •Sarah Dekat ,NCSS 2006; Radioimmunoassay (RIA): A Remarkably Sensitive Bioassay 34
  35. 35. THANK YOU… 35