RADIO IMMUNO ASSAY

This presentation explains about the Immunoassay ,radio immuno assay, definition, types, Principle , procedure, steps involved ,advantages ,disadvantages ,Application, RIA in insulin. RIA in Digitalis drug ligand etc....

1. RADIOIMMUNO ASSAY PRESENTED BY, SURIYAPRIYA .K, 1 st yr M.PHARM DEPARTMENT OF PHARMACEUTICAL ANALYSIS, ADVANCED PHARMACEUTICAL ANALYSIS, KMCH COLLEGE OF PHARMACY, COIMBATORE. KMCH COLLEGE OF PHARMACY 1 7/20/2021

2. OVERVIEW IMMUNOASSAY ANTIBODY ,ANTIGEN, ANALYTE RADIOIMMUNOASSAY INTRODUCTION HISTORY PRINCIPLE REQUIREMENTS FOR ASSAY STEPS INVOLVED IN RIA PROCEDURE INVOLVED IN RIA INSTRUMENTATION RIA IN DIGITALIS RIA IN INSULIN MERITS OF RIA DEMERITS OF RIA DRAWBACK REFERENCE 7/20/2021 KMCH COLLEGE OF PHARMACY 2

3. IMMUNO ASSAY An immunoassay is a biochemical test ,that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody or an antigen. An immunoassay is a test that uses antibody and antigen complexes . An antibody and antigen complex is also known as an immune- complex . refers to an immune response that causes the body to generate antibodies . refers to a test . “ Immuno” “ Assay ” 7/20/2021 KMCH COLLEGE OF PHARMACY 3

4. RADIOIMMUNO ASSAY COUNTING IMMUNOASSAY ENZYME LINKED IMMUNO ASSAY (ELISA) FLUOROIMMUNOASSAY CHEMILUMINESCENCE IMMUNOASSAY TYPES OF IMMUNO ASSAY 7/20/2021 KMCH COLLEGE OF PHARMACY 4

5. ANTIBODIES, ANTIGENS AND ANALYTES  An antibody is a protein that is produced by the body immune response to an “invading” (foreign) substance.  Antibodies are produced as part of the body’s immune response to protect itself.  An antigen is the substance that the body is trying to “fight off” by mounting an immune response. For example, D rug is the antigen that binds to the antibody.  An immunogen is a substance that elicits immune response. E.g. drug-protein conjugate. 7/20/2021 KMCH COLLEGE OF PHARMACY 5

6. An analyte is anything measured by a laboratory test. In immunoassay testing, the analyte may be either an antibody, or an antigen. Immunoassays utilize one or more selected antibodies to detect analytes of interest. The analytes being measured may be:- 1. That are naturally present in the body (such as a thyroid hormone) 2. The body produces but are not typically present (such as a cancer antigen) 3. Do not naturally occur in the body (such as an abused drug)  TO BE CONTINUED , 7/20/2021 KMCH COLLEGE OF PHARMACY 6

7. STRUCTURE OF ANTIBODIES Antibodies are produced by the B lymphocytes , Y shaped (Immunoglobulin ) The most important one is immunoglobulin G (IgG). IgG is a protein composed of two main structural and functional regions: Fab region : Contains the antigen (Ag) binding site that varies between different antibodies. Fc region: Region of constant structure within an antibody class. 7/20/2021 KMCH COLLEGE OF PHARMACY 7

8. RADIOIMMUNO ASSAY Radioimmunoassay is a t echnique for determining antibody levels by introducing an antigen labelled with a radioisotope and measuring the subsequent radioactivity of the antibody component. For example,  H ormone  levels in  blood (insulin ) by use of  antibodies . The RIA technique is extremely  sensitive  and extremely  specific . It is the technique used for the estimation of any ligand / antigen in biological fluids ,based on antigen antibody reaction . 7/20/2021 KMCH COLLEGE OF PHARMACY 8

9.   HISTORY Developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma. Rosalyn Yalow   was a nuclear physicist. She  developed  radioimmunoassay (RIA) together with doctor  Solomon Berson It represented the first time that hormone levels in the blood could be detected by an in vitro assay. In 1977 Yalow received the Nobel Prize for her and Berson’s development of RIA 7/20/2021 KMCH COLLEGE OF PHARMACY 9

10. REQUIREMENTS   Micro titer plate / Polypropylene Test tubes Pure antigen Pipette Radio labelled antigen Antibodies Standard’s / Reference Vortex mixer Centrifuge Radioactive counter 7/20/2021 KMCH COLLEGE OF PHARMACY 10

11. RADIO IMMUNO ASSAY TWO METHODS EMPLOYED FOR DRUG DETECTION IN BIOLOGICAL MATRICES WITH DOUBLE ANTIBODY RIA WITH COATED TUBE RIA 7/20/2021 KMCH COLLEGE OF PHARMACY 11

12. PREPARATION AND RADIOLABELING OF THE ANTIGEN Antigens prepared by  synthesis of the molecule  isolation from natural sources Radiolabeling [Tagging procedure ]  3H,14C,125I are used as radioactive tags  Antigens are tagged to 3H , 14c , 125I  Tagging should NOT affect antigenic specificity and a ctivity 7/20/2021 KMCH COLLEGE OF PHARMACY 12

13. PREAPRATION OF SPECIFIC ANTIBODY Antibody can be either polyclonal or monoclonal . Antigen injected intradermally into rabbit or guinea pigs antibody production . Antibodies recovered from the serum . Some ligands are not antigenic. With the low molecular weight substances such as the thyroxine, steroid hormones, and drugs conjugation is essential . . 7/20/2021 KMCH COLLEGE OF PHARMACY 13

14. POLYCLONAL ANTIBODY MONOCLONAL ANTIBODY Cheap to produce Expensive to produce Mixed population of antibodies Single antibody species May bind to different areas of the target molecules Will only bind single specific site Tolerant of small changes in protein structure May recognise a particular protein form Monoclonal antibody Polyclonal antibody 7/20/2021 KMCH COLLEGE OF PHARMACY 14

15. PRINCIPLE OF RIA : The basic principle of radioimmunoassay is competitive binding, where a radioactive antigen ("tracer") competes with a non-radioactive antigen for a fixed number of antibody or receptor binding sites.  The radio labeled antigen should have similar biological activity and immunogenicity like that of native antigen . LABELED ANTIGEN + SPECIFIC ANTIBODY LABELED ANTIGEN Ag *ANTIBODY complex Ag* Ab Ag*Ab UNLABELED ANTIGEN Ag UNLABELED ANTIGEN - ANTIBODY COMPLEX Ag Ab 7/20/2021 KMCH COLLEGE OF PHARMACY 15

16. TO BE CONTINUED , The free antigen and labelled antigen are separated and washed out . The radioactivity of the labeled antigen antibody complex is measured using scintillation counter or gamma counter. The measured radioactivity is inversely proportional to the concentration of antigen . More is the concentration of antigen , lesser is the radioactivity of the complex and vice versa . 7/20/2021 KMCH COLLEGE OF PHARMACY 16

17. CALIBRATION CURVE : From these data, a standard binding curve, like the one shown in red, can be drawn. The samples to be assayed (the unknowns) are run in parallel .  After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve. 7/20/2021 KMCH COLLEGE OF PHARMACY 17

18. STEP FOLLOWED IN RADIOIMMUNOASSAY 7/20/2021 KMCH COLLEGE OF PHARMACY 18

19. PREPARATION AND CHARACTERIZATION OF ANTIGEN The antigen whose concentration is to be measured , should be available in pure form. Also required to create antibodies ,in assay process to form complex . RADIO LABELING OF ANTIGEN TAGGING , where antigen is tagged with radio active elements like 125 I , 14 C PREPARATION OF SPECIFIC ANTIBODY Specific antibodies are produced by injecting the antigen in mouse , rabbit, guinea pig & collect & purify the antibodies . In case of drug or other compounds (Eg . morphine , hormones ) which are not antigenic . They combined with albumin , to become antigenic . DEVELOPMENT OF ASSAY VALIDATION OF METHOD Parameter like sensitivity ,specificity, precision, linearity etc 7/20/2021 KMCH COLLEGE OF PHARMACY 19

20. PROCEDURE INVOLVED IN RADIO IMMUNOASSAY 7/20/2021 KMCH COLLEGE OF PHARMACY 20

21. THE ASSAY IS CARRIED OUT IN WELL PLATES (TYPICALLY 96 WELL PLATE ) Where inner surface is coated with antibodies RADIO ACTIVE ANTIGEN IS ADDED TO ALL THESE WELLS Which forms a complex with antibodies THE UNLABELED ANTIGEN OF KNOWN CONCENTRATION AND ANTIGEN OF UNKNOWN CONCENTRATION (SAMPLE) Where known conc. Added to multiple wells and unknown conc. Added to one of the well COMPETITION BETWEEN LABELED AND UNLABELED ANTIGEN TAKE PLACE Due to limited binding sites present in antibodies RADIO LABELED ANTIGEN BOUNDED TO ANTIBODY Separated by precipitation/chromatography/gel filtration 7/20/2021 KMCH COLLEGE OF PHARMACY 21

22. ANTIBODY COMPLEX FORMED This leads to displacement of radio labeled antigen by unlabeled antigen AFTER INCUBATION TIME THE PLATE ARE REMOVED AND WASHED This remove free antigen and unbound antigen GAMMA OR SCINTILLATION COUNTER Measuring the radio activity MORE CONC.OF FREE ANTIGEN LESSER RADIO ACTIVITY IN COMPLEX FROM THE GRAPH OF RADIO ACTIVITY VS CONC.THE UNKNOWN ,CONC.OF ANTIGEN CAN BE DETERMINED 7/20/2021 KMCH COLLEGE OF PHARMACY 22

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24. Figure  illustrates how a limited amount of antibody binding sites contained in a test tube or microplate well can either bind unlabeled ligand (in this example, Hepatitis B Surface Antigen) or radiolabeled ligand. As the amount of unlabeled ligand increases, there is consequently less radiolabeled ligand bound. The unlabeled ligand can come from either a “calibration standard” or the sample that you are trying to measure. 7/20/2021 KMCH COLLEGE OF PHARMACY 24

25. SEPARATION TECHNIQUE To separate free from bound labelled antigen separation techniques are : a ) physical methods :-filtration ,chromatography ,electrophoresis, charcoal dextran adsorption etc. b) Chemical methods :-organic solvents such as ethanol, dioxane , PEG, ammonium sulfate for precipitating antibody . C) Immunological precipitation :- 2 nd antibody d) Solid phase systems :- with antibodies on solid support or matrix 7/20/2021 KMCH COLLEGE OF PHARMACY 25

26. INSTRUMENTATION The following steps are involved in RIA: Centrifuge Radioactive counter 7/20/2021 KMCH COLLEGE OF PHARMACY 26

27. swing- bucket rotor  CENTRIFUGE Fixed angle head rotor generating 1200-2500 rpm  generating 3500-4000 rpm  pellet is formed at an angle pellet is formed at the bottom of the test tube T he latter type is preferred T he former type is preferred 7/20/2021 KMCH COLLEGE OF PHARMACY 27

28. RADIOACTIVE COUNTER Gamma counters Scintillation Counters gamma-energy emitting isotopes , for instance :  125 I-the more common iodine-isotope.   beta-energy-emitting isotopes , such as : tritium  3 H and  14 C-(Carbon-14) isotopes . 7/20/2021 KMCH COLLEGE OF PHARMACY 28

29. GAMMA COUNTER scintillation Counter 7/20/2021 KMCH COLLEGE OF PHARMACY 29

30. RIA OF DIGITALIS Digitalis Commonly known as Foxglove leaves, Generic name – Digitalis purpurea Family - Scrophulariaceae. It is also used for drug preparations that contain cardiac glycosides, like Digoxin & Digitoxin extracted from various parts of the plant. USES : Increase cardiac contractility Antiarrhythmic agent Atrial flutter Atrial fibrillation 7/20/2021 KMCH COLLEGE OF PHARMACY 30

31. PRINCIPLE OF RIA FOR DIGITALIS The assay is based on the use of 125-iodine-labelled digoxin and of a gel equilibration technique for the separation of antibody- bound and free digoxin. Digoxin in serum samples competes with radio-labelled (125-1) digoxin derivative for binding sites on the antibody to digoxin. The unbound digoxin is then separated from bound form. It is then quantified by counting radioactivity & concentration of unlabeled digoxin in serum sample is calculated by comparison to digoxin standard . 7/20/2021 KMCH COLLEGE OF PHARMACY 31

32. Digoxin Standards - From 0.5- 8.0 ng/ml Anti-serum and Tracer solutions Digoxin antibodies raised in rabbits by subcutaneous I njections of digoxin-bovine serum albumin Phosphate saline buffer Digoxin Radio Immuno assay kit, with 3-0-succinyl D igoxigenin tyrosine 125-1. Materials Used For Assay 7/20/2021 KMCH COLLEGE OF PHARMACY 32

33. The assay is carried out using 3-O-succinvl digoxigenin tyrosine 125-Ⅰ. 1 ml of phosphate buffer solution + 0-50 µl of standard digoxin solution + 10 µl of 3-0-succinyl digoxigenin tyrosine 125-Ⅰ Add 10 µl of digoxin anti-serum, all contents are mixed well. Standard Curve PROCEDURE FOR ASSAY 7/20/2021 KMCH COLLEGE OF PHARMACY 33

34. To 50µl of patient's plasma, + 1 ml of phosphate buffered saline. To this add 10µ of labelled solution, & 10µl of digoxin anti-serum, A ll contents are mixed well All the tubes are allowed to stand and 0.5ml of charcoal solution is added to all tubes. The tubes are then centrifuged, gamma radioactivity. Procedure For Unknowns 7/20/2021 KMCH COLLEGE OF PHARMACY 34

35. RIA measurements of insulin RIA measurements of insulin combined with glucose estimations. Can diagnose hyperinsulinism due to insulin secreting tumors of the pancreas. Insulin (in micro units per ml) to glucose (in mg/100 ml) ratios of more than 0.30 is indicative of this condition. Measurement of insulin and C-peptide concentrations following arginine stimulation Test . In this test, 300 ml of a 10% solution of arginine is infused into a fasting patient who has not had insulin, over a period of 30 minutes and blood taken at - 0, 15, 30, 45, 60 and 90 minutes. 7/20/2021 KMCH COLLEGE OF PHARMACY 35

36. In Type I, insulin dependent diabetes, basal levels of C-peptide would be less than 0.3 ng/ml and there would be no response to arginine. If basal C-peptide is low, say 0.3-1.0 ng/ml, and there is some response to arginine (a 100% rise), the patient is still insulin deficient. In typical Type II diabetes, basal levels would be 2.5 ng/ml or over, and a 100% rise on stimulation would be seen. RIA for microalbuminuria is becoming popular as a predictor of the development of clinical nephropathy in diabetics. TO BE CONTINUED , 7/20/2021 KMCH COLLEGE OF PHARMACY 36

37. MERITS OF RADIOIMMUNOASSAY High specificity and hence there is no interference from other substances. High sensitivity – Immuno reaction are very sensitive , so when antibody with high affinity are used , it is possible to detect antigen up to a few picogram (10-12 g). High precision and accuracy Applicable to wide variety of compounds in various fields pharmacology Endocrinology Oncology Epidemiology Clinical immunology 7/20/2021 KMCH COLLEGE OF PHARMACY 37

38. DEMERITS OF RADIOIMMUNOASSAY Expensive compared to other methods ,as radioactive materials are used . Radiation hazards because of Usage of 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. Development of specific antibodies to the antigen . . Compliant technique than other methods compared to Tlc , Hptlc , etc.. 7/20/2021 KMCH COLLEGE OF PHARMA 38

39. APPLICATION OF RADIO IMMUNOASSAY The test can be used to determine very small quantities (e.g. nanogram) of antigens and antibodies in the serum. The test is used for quantitation of hormones, drugs(Digitoxin or digoxin ), HBsAg in blood ,HIV 1& 2 Virus and other viral antigens. Analyze nanomolar and picomolar concentrations of hormones in biological fluids. Certain abused drugs , Eg . MORPHINE Ria has been used to assay plasma levels of : 1. M ost of hormones 2.Insulin in human plasma 3.Beta - HCG in females 7/20/2021 KMCH COLLEGE OF PHARMACY 39

40. DRAWBACKS : The cost of equipment and reagents are expensive . Short shelf life of radiolabeled compounds . The problems associated with the disposal of radioactive waste. H azards if preparing and handling the radioactive antigen. Both 125I or 131I emit gamma radiation that requires special counting equipment . 7/20/2021 KMCH COLLEGE OF PHARMACY 40

41. THE SPECIFICITY OF AN RIA IS IMPROVED BY: ( a) The use of pure immunogens or conjugates. (b) Pure labelled compounds. (c) Pure analyte for assay, pre-extracted and purified if needed. (d) Immunization with specific subunits, e.g. b-hCG (e) Saturation with cross reactants, e.g. TSH antibody with hCG (f) Use of monoclonal antibodies of high specificity 7/20/2021 KMCH COLLEGE OF PHARMACY 41

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43. REFERENCE https://microbenotes.com/radioimmunoassay-principle-uses-and-limitations/ https://www.google.com/search?q=radio+immunoassay&oq=&aqs=chrome.2.69i59i450l8.1354187j0j7&sourceid=chrome&ie=UTF-8# Text book of instrumental analysis by the author Ravi shanker https://www.brainkart.com/article/Radioimmunoassay--Instrumentation_30954/ The production of antibodies for radioimmunoassay ; https://pubmed.ncbi.nlm.nih.gov/772533 Journal of Perkin elmerhttps://www.perkinelmer.com/lab-products-and-services/application-support-knowledgebase/radiometric/radioimmunoassays.html https://www.amjmed.com/article/0002-9343(71)90237-3/fulltext 7/20/2021 KMCH COLLEGE OF PHARMACY 43