Radioimmunoassay (modified copy)

Meghana C

1. RADIOIMMUNOASSAY PRINCIPLE AND INSTRUMENTATION Presenter MEGHANA C II M.Sc Med Biochemistry Dept. Of Biochemistry JSS Medical College Mysuru Guide Dr ABHIJITH D Assistant Professor Dept of Biochemistry JSS Medical College Mysuru

2. CONTENTS HISTORY INTRODUCTION PRINCIPLE INSTRUMENTATION REQUIREMENTS OF RIA METHODOLOGY OF THE ASSAY PROCEDURE ADVANTAGES DISADVANTAGES APPLICATIONS

3. HISTORY Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens by use of antibodies RIA was primarily developed by Solomon Berson & Rosalyn Yalow(1959) for the quantitative measurement of insulin in human plasma.

4. In 1977 Yalow received the Nobel Prize in Physiology or Medicine for her and Berson’s development of RIA as it applied to tracing hormones in the body.

5. Rosalyn Yalow in the laboratory. She conducted her research at the VA Medical Center in Bronx, New York, from 1947 to 1991 .

6. Since Nobel Prizes are only given to the living, Yalow received the award without Berson , who died in 1972. She shared it with two other scientists, who were honored for their work on hormone production in the brain. Rosalyn Yalow receives the Nobel Prize in Physiology or Medicine 1977 from King Carl XVI Gustaf of Sweden.

7. INTRODUCTION “ 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.

8. CATEGORIES OF IMMUNOASSAY TESTS Competitive Non-competitive Homogeneous Heterogeneous

9. “RADIOIMMUNOASSAY IS A TECHNIQUE FOR DETERMINING ANTIBODY LEVELS BY INTRODUCING AN ANTIGEN LABELED WITH A RADIOISOTOPE AND MEASURING THE SUBSEQUENT RADIOACTIVITY OF THE ANTIBODY COMPONENT”.

10. RIA is specific, sensitive, & rapid assay technique. The major disadvantage of RIA is health & safety risks by the use of radiation & maintaining licensed radiation & disposal program is difficult.

11. The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g., Drug detection from blood samples Blood bank screening for contaminants,e.g. The hepatitis virus Detection of cancer biomarkers Measurement of hormone levels Measurement of disease associated antigen levels Measurement of vitamins or other proteinaceous antigen level. Diagnosis of allergies

12. OBJECTIVE The objective is to determine the concentration of a unlabelled antigen. So in order to conduct RIA a standard curve is made with concentration versus % of radioactive compound bound.

13. STANDARD CURVE OF RIA

14. PRINCIPLE Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity.

15. PRINCIPLE THE TECHNIQUE The Principle of Radioimmunoassay (RIA) 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)

16. 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. serum A Antigen Anti A Antibodies 2. Radiolabeled A antigen 3. Unlabeled A antigen Radioisotopes 125 I Tritium

18. 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. The radioactivity of each is measured.

19. INSTRUMENTATION REQUIREMENTS Microtiter plate/ test tube Pure antigen Radio labelled antigen Anti body Standards Centrifuge Radio active counter

20. INSTRUMENTATION MICRO TITER PLATE  :- A micro titer plate is used mostly used for this assay. A microtiter plate could have 6,24,96,384 or even sometimes 1536 wells arranged in rows.Each well of a microplate can only hold very small amount of liquid. PURE ANTIGEN  :- Antigen may be obtained from biological sample or by synthetic form it should be pure. It is used as standard or calibrator. RADIO LABELLING OF ANTIGEN  :- The most commonly used radiolabels in RIA are tritum and iodine.They have adequate activity and have long enough half lifes .

21. INSTRUMENTATION ANTIBODY  :- specific antibodies are obtained by injecting the Ag to animals. STANDARDS  :- % antibody bound with antigen in unknown sample Amount of antigen in unknown sample

22. INSTRUMENTATION CENTRIFUGE  :- Used for the separation of precipitated and supernatant liquid from the sample.

23. RADIOACTIVE COUNTER

24. INSTRUMENTATION RADIO ACTIVE COUNTERS  :- Two types of counters are used namely, 1.Gamma counter 2. Scintillation counters Gamma counter:- These are used for the gamma energy emitting isotopes E.g. 125 I . Scintillation counter :- These are used for counting beta energy emitting isotopes E.g. Tritium 3 H & 14 C Isotopes

25. REQUIREMENTS OF RIA

26. REQUIREMENTS OF RIA PREPARATION & RADIOLABELLING OF THE AG Antigens prepared by, Synthesis of the molecule Isolation from natural sources

27. 2. RADIOLABELLING [TAGGING PROCEDURE] 3 H , 14 C, 125 I are used as radioactive tags Antigens are tagged to 3 H , 14 C, 125 I NOTE: Tagging should NOT affect Antigenic specificity & Antigenic activity !

28. REQUIREMENTS OF RIA 3. PREPARATION OF THE SPECIFIC ANTIBODY Antigen injected intradermally into rabbits or guinea pigs to induce antibody production Antibodies recovered from the serum

29. REQUIREMENTS OF RIA 4. DEVELOPMENT OF THE ASSAY SYSTEM Crucial step is separation of unbound antigens Ab 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 Ag

30. METHODOLOGY OF THE ASSAY In RIA the sequential steps followed are: Mix a fixed concentration of antiserum containing specific antibody with a constant quantity of radiolabelled antigen. Incubate it for specified duration & at an appropriate temperature usually +4°c. A definite volume of sample containing unlabelled antigen to be measured is added to the reaction test tube.

31. METHODOLOGY OF THE ASSAY The antibody reacts with both radioactive & unlabelled antigen forming an ab-radiolabelled antigen complexes. Both radioactive & unlabelled antigens are more or less same immunochemically, they will compete for limited number of antibody sites available. The radio activity falls because the unlabelled antigen dilutes i.e.)reducing the no of labelled antigen combining with antibody. The counts obtained from the radioactivity are used to determine the unlabelled antigen concentration in the sample, the interpretation being done on the standard curve.

32. PROCEDURE A specific quantity of antigen is made radioactive. 2. The radiolabeled antigen is mixed with a specified amount of antibody for a particular antigen causing the two to bind to each other. 3. A serum sample from a patient that contains an unknown quantity of the same antigen is added causing the unlabeled antigen from the serum to compete with the radiolabeled antigen for the antibody binding sites.

33. PROCEDURE 4. The unlabeled antigen concentration increases causing it to bind more to the antibody. 5. The radiolabeled variant is displaced reducing the ratio of antibody-bound radiolabeled antigen. 6. The bound antigen separates from the unbound ones. 7. The remaining free antigen’s radioactivity in the supernatant is measured.

34. PROCEDURE Microtiter plate is taken Coated with anti A monoclonal antibodies Radiolabeled antigens are added to the well in excess quantity No antibody is left free ; some radiolabeled antigens are left unbound

35. Unbound antigens are removed with the help of washing Now Radioactivity in the antibody of the well is 100% A very small but known amount of unlabeled antigen is added This addition will create competition between radio labeled & unlabeled antigen Few radio labeled antigens are removed from the antibodies Addition of unlabeled antigen Radiolabeled Ags decreased Due to the addition of unlabeled Ag

36. 0 ng 100% 1 ng 90% 2 ng 80% 3 ng 70% 4 ng 60% 5 ng 50% 6 ng 40% 7 ng 30% 8 ng 20% 9 ng 10% 10 ng 0%

37. Serum sample A Ag...? Microtiter plate 40% Radioactivity Ans :- 6ng

38. 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 reusable

39. DISADVANTAGES Radiation hazards: Uses radio labelled reagents Requires specially trained persons labs require special license to handle radioactive materials Requires special arrangements for, Requisition, handling, Storage of radioactive material Radioactive waste disposal. 5. Expensive instrumentation for the conting of radioactivity, Both 125 I or 131 I emmit gamma radiation that requires special counting equipment.

40. APPLICATIONS Analysis of hormones, vitamins, metabolites, diagnostic markers Examples :- ACTH, FSH, T3, T4, Glucagon, Insulin , Testosterone vitamin B12, prostaglandins, glucocorticoids , 2. Therapeutic drug monitoring: Examples :- Barbiturates, morphine, digoxin , 3. Diagnostic procedures for detecting infection Examples :- HIV, Hepatitis A, B etc

41. 4. Used to assay Plasma levels of: Most of our hormones; Digitoxin or digoxin in patients receiving these drugs; Certain abused drugs Screening donated blood Hepatitis B Hepapitis C HIV

42. 5. In Endocrinology Insulin, HCG, Vasopressin Detects Endocrine Disorders Physiology of Endocrine Function 6. In Pharmacology Morphine Detect Drug Abuse or Drug Poisoning Study Drug Kinetics

43. 7. Clinical Immunology Antibodies for Inhalant Allergens Allergy Diagnosis 8 . Early Cancer Detection and Diagnosis 9. Narcotic drug detection 10. Tracking of leukemia virus 11. Diagnosis and treatment of peptic ulcers 12. Measuring “rheumatoid factors” and other auto antibodies in autoimmune disease likeluous erythematosus .

44. RECENT APPLICATIONS Refinement of zalcitabine pharmacokinetics Estradiol measurement in translational studies of breast cancer. Significance of prostate specific antigen in prostate cancer patients & in non cancerous prostetic disease patients.

45. REFERENCES Yallow R, Berson . Immunoassay Of Endogeneous Plasma Inslin In Man. J.Clin . Iinvest 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