Radioimmunoassay ppt by dr Saurabh maurya

Radioimmunoassay

1. Radioimmunoassay Dr. Saurabh Maurya Junior Resident – 3 Department of Pharmacology and Therapeutics King George’s Medical University Lucknow (U.P.) India Email- [email protected]

2. Content : Introduction Principle Procedure Applications Advantage Disadvantage Summary

3. Introduction: Immunoassay B iological tests used to detect the presence or concentration of a specific chemical, such as toxins or hormones It uses antigen-antibody complex It became very popular due to its high sensitivity and specificity

4. Enzyme immunoassay Fluoro immunoassay Luminescence immunoassay Radio immunoassay

5. Radioimmunoassay Radioimmunoassay (RIA) used to measure concentration of antigen by use of antibody with the help of radioisotopes Rosalyn Yellow and Solomon Berson got Nobel price in 1977 for discovery of RIA Extremely sensitive and specific Eg . Radio Allergo Sorbant Test (RAST) Immuno Ag-Ab binding theory Radio Use of radio active material Assay Detection of compounds

6. Labelled Antigen : Antigen of interest are labelled with radioactive substance Gamma-emitting isotopes such as 125 I Beta emitting isotopes such as 3 H ( titrium ) Sample Antigen : Unlabelled antigen or test antigen which is to be detected

7. Primary antibody Specific antibody that binds to the antigen Can’t differentiate between labelled or sample antigen Secondary Antigen Binds to primary antibody It is PEGylated to facilitate the separation process

8. Principle : Immune reaction : Antigen – antibody binding Competitive binding or competitive displacement reaction Measurement of radio emission

9. Requirement: Specific antiserum to the antigen to be measured Availability of a radioactive labeled form of antigen A method in which the antibody-bound antigen can be separated from unbound antigen An instrument to count radioactivity

10. Procedure: Radioactive antigen Primary antibody Add unlabeled antigen Radioactive antigen displaced by Unlabeled antigen Precipitate Ag-Ab complexes with secondary antibody Radioactivity of supernatant = free antigen Radioactivity of precipitate = bound antigen Secondary antibody

11. Procedure : Ag* + Ab All Ag binding site of Ab get saturated Test Ag added Both Ag & Ag* competes for binding site As the concentration of test Ag increase m ore labelled Ag* will be d isplaced from binding site

12. Ag-Ab complex + Ag*+ Ab Centrifugation/Secondary antibody Radioactivity of supernatant and precipitate i s measured by gamma counter Continued…

13. Variable Quantity Radioactivity of of unlabeled Ag Ag*Ab complex 0 ng 100 % 1 ng 90 % 2 ng 80 % 3 ng 70 % 10 ng 10 %

14. Application : Diagnostic purpose Hormone measurement like T3 T4 , growth hormone, insulin Tumor marker analysis like PSA , AFP , hCG Cardiac marker like t ropoin , CK-MB Infectiuos diseases like Ag related to HIV, Hep . B

15. Role of RIA in drug monitoring Detecting drugs for substance abuse like morphine, amphetamine Detecting prohibited drugs in sports To measure drug level to ensure therapeutic efficacy To measure drug level for toxicity

16. In Pharmacology and drug discovery 1. Pharmacokinetic studies Absorption, distribution, metabolism, excretion of drugs 2. Drug concentration measurement It can quantify drug levels in preclinical and clinical studies during drug development and drug optimization 3. Bioavailability and metabolite analysis

17. Endocrine and reproductive medicine: Fertility testing eg . LH, FSH level Pregnancy testing Assessment of gonadal function by measuring hormones like testosterone and sex hormone binding globulin (SHBG)

18. In research and life sciences Protein quantification Environmental analysis Food and beverage industry Veterinary medicine

19. Advantage: High Sensitivity Radioactivity is easily measured by gamma counter. High affinity It can detect up to 10-12 pg of antigen High Specificity Antigen-antibody reaction

20. It is a saturation – displacement analysis which increases reproducibility Can be used for variety of samples like saliva, blood, urine, serum

21. Disadvantage : Expensive Special arrangement for waste disposal is required Need trained technical staff to handle Use of radioactive materials make cause problem in accidental intake Special protection is required during handling

22. Summary Radioimmunoassay is highly sensitive and highly specific Competitive displacement reaction is the main principle Radioactivity of precipitate and supernatant is measured RIA is used for various diagnostic, research and drug discovery purpose

23. References: Yalow RS, Berson SA. Immunoassay of endogenouscause harm cial protec -plasma insulin in man. J Clin Invest. 1960;39:1157-75 . Odell WD, Daúghadsy WH (Eds.). Principles of Competitive Protein Binding Assays. Philadelphia: I.B. Lippincott Co.; 1971 Rodbsd D, Rayford PL, Cooper IA, Ross GT. Statistical quality control and routine data processing for radioimmunossssys (RIA) and immunoradiometric asssays (IRMA). Clin Chem. 1974;20:1255-70.5. Adams JM, et al. Zalcitabine population pharmacokinet-ics : application of radioimmunoassay. Antimicrob . Agents Chemother . 1998;42(2)409-13. doi:10.1128/AAC.42.2.409 Lonning PE. Estradiol measurement in translational studies of breast cancer, Part A. Steroids. 2015;99:26-31.ISSN: 0039 - 128X; https:// doi . org/10. 1016/ j.steroids.2014.08.008 (http://www.sciencedirect.com/sci-ence/article/pii/S0039128X14002013 ). Szeto A, McCabe PM, Nation DA, et al. Evaluation of enzyme immunoassay and radioimmunoassay methods for the measurement of plasma oxytocin. Psychosom Med. 2011;73(5):393-400. doi:10.1097/PSY.0b01331821df0c2.Epub 2011 Jun 2. PMID: 21636661; PMCID: PMC3118424.8. Hanif M, et al. Significance of prostate specific antigen in prostate cancer patients and in non cancerous prostatic disease patients; 1 Pak Med Assoc. 2007;57(5)248-51

24. THANK YOU

25. Questions: What is labelled and unlabelled antigen? What are the principles of RIA? What are the requirements for RIA? What are the diagnostic use of RIA? What are the advantages of RIA?