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Radio immuno assay is an elegant technique.it includes the principles,reagents,and the application.also some recent applications

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  1. 1. Radioimmunoassay Presented by : Justin K Joy First Year M.Pharm Pharmaceutical Analysis St.James college of Pharmaceutical Sciences.
  2. 2. Immunoassay • An immunoassay is a test that uses antibody and antigen complexes • An antibody: antigen complex is also known as an immune- complex • “Immuno” refers to an immune response that causes the body to generate antibodies • “assay” refers to a test • immunoassay is a test that utilizes immuno complexing when antibodies and antigens are brought together Radio immuno assay 2
  3. 3. Antibodies, Antigens and Analytes  An antibody is a protein that is produced by the body in 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, the drug is the antigen that binds to the antibody.  An immunogen is a substance that elicits immune response. E.g. drug-protein conjugate. Radio immuno assay 3
  4. 4. Antibodies, Antigens and Analytes  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) Radio immuno assay 4
  5. 5. Structure of Antibodies • Antibodies are produced by the B lymphocytes. • The most common one is immunoglobulin G (IgG). • IgG is a protein composed of two main structural and functional regions: Radio immuno assay 5 Fab region: Contains the antigen (Ag) binding site that varies between different antibodies. Fc region: Region of constant structure within an antibody class.
  6. 6. Radioimmunoassay  Radio Immuno Assay (RIA) is an elegant tech. in analytical chemistry.  If substance to be analysed is in very low quantities, in the orders of micrograms, nanograms, conventional methods like gravimetric and colorimetric method fail.  RIA finds extensive application in the assay of many substances which are present in trace amount in blood. Radio immuno assay 6
  7. 7. Radio immuno assay 7 History Developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma. 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
  8. 8. Principle Of RIA • The amount of Ab per tube is kept constant, the amount of antigen added (known or unknown) is the variable parameter. • The added antigen will be distributed between a bound (B) and a free (F) fraction. • This distribution is governed by the association constant (KA) of the Ab: Ab + Ag  AgAb K = [AbAg] /[Ab][Ag] Radio immuno assay 8
  9. 9. Principle Of RIA Competitive binding of radiolabelled antigen and unlabelled antigen to a high affinity antibody. The labelled antigen is mixed with the antibody at a concentration that saturates the antigen –binding sites of the antibody. As the concentration of the unlabelled antigen increases more labelled antigen will be replaced from the binding site. Radio immuno assay 9
  10. 10. Radio immuno assay 10 The decrease in the amount of radiolabelled antigen bound specific antibody in the presence of the test samples is measured to determine the amount of antigen Present in the test sample. In std Condition, amount of labelled antigen bound to the antibody decreases as the amount of unlabelled antigen increases in sample. Antibody Unlabeled antigen Labeled antigen
  11. 11. Reagents used in RIA: 1. A tracer i.e. a labelled ligand. 2. A binder (Antibody) which is the specific antiserum. 3. A separation system to separate to separate the ‘bound’ and ‘free’ phases. 4. A standard (in highly pure form) 5. A free human antiserum. Radio immuno assay 11
  12. 12. Reagents used in RIA: Tracer The radioisotopes used are – Beta emitters- 3H and 14C Gamma emitters- 125I Radio immuno assay 12
  13. 13. Reagents used in RIA: Tracer Tritium – 3H:  Weak β− ray emitter  Significantly lower energy than 14C  Long physical half-life of 12.3 yrs  Biological half-life: 10 – 12 days  Produced by neutron bombardment of a lower hydrogen isotope  Used for drugs like proteins and amino acids Radio immuno assay 13
  14. 14. Reagents used in RIA: Tracer Carbon-14:  Weak β− ray emitter  Long physical half-life (~ 5.7 x 103 yrs)  Biological half-life: Bound – 12days; Unbound – 40days  Commercially available as Barium carbonate-14C Radio immuno assay 14
  15. 15. Reagents used in RIA: Tracer Iodine-125:  Low γ−emission: 35.4 keV  High specific activity  Short physical half-life: 60 days Radio immuno assay 15
  16. 16. Reagents used in RIA: Tracer  Positron vs. Gamma Isotopes:  The positron (β-emitting) radionuclides are mainly restricted for in-vitro experiments.  The γ-emitting radionuclides are useful for in-vivo imaging Other commonly used isotopes:  Positron: 11C, 13N, 15O, and 18F.  Gamma : 111In(indium), 123I, 131I, 153Sm(Samarium), 75Se. Radio immuno assay 16
  17. 17. Reagents used in RIA: Tracer  125I is most favoured because- • It can be obtained with specific activity and almost 100% isotopic abundance, thus reducing counting time and being economic. • Convenient half-life (60.2 days) hence shelf life for labelled antigen is long. • Iodine is natural constituent of thyroxine and tri- iodothyronine. • It can be easily introduced into peptide molecules, steroids. • Gamma emission permits the use of simple inexpensive equipment for counting radioactivity. Radio immuno assay 17
  18. 18. Reagents used in RIA: Tracer  Disadvantages- o The damage to the ligand may occur during storage. o Health hazards are more. Radio immuno assay 18
  19. 19. Reagents used in RIA: Tracer 3H is more efficient when relatively small sample is to be determined.  Advantages- • Long shelf life (12.3 yrs). • Higher affinity and no necessity for derivative preparation. • Minimal health hazards.  Disadvantages- • Requires scintillation counter which is costly • Low specific activity (10000 14C = 100 3H = 1 125I). Radio immuno assay 19
  20. 20. Radio immuno assay 20 Specific antiserum(binder/antibody) It is prepared by injecting (repeatedly) the antigen together with Freund’s adjuvant into suitable animal such as guinea pig, rabbit or goat. Molecule like thyroid hormone,steroids,drug are not immunogenic. So they are conjugated to carrier proteins and polymer to make them immunogenic
  21. 21. Radio immuno assay 21 SEPERATION SYSTEM It is required because the bound fraction does not precipitate spontaneously at the low concentration. Variety of procedure are available a)Physical method Filtration,chromatography,electrophoresis,charcoal dextran adsorption  b)Chemical method-Organic solvent such as ethanol,dioxane,PEG or salts such as sodium ,zinc and ammonium sulphate.  C)Solid phase system
  22. 22. Radio immuno assay 22 A STANDARD ie LIGAND (ANALYTE) IN HIGHLY PURE FORM Drugs ,protein,hormone etc must be in pure form so they can be diluted . Standard are prepared in ligand free serum In case of protein ,hormones the standard should be prepared preferably with the hormone from the species from which the serum is going to be analysed.
  23. 23. Radio immuno assay 23 LIGAND FREE HUMAN SERUM It is prepared by treating human serum with charcoal . It can also be prepared by collecting serum from volunteers in whom production of that ligand or hormone has been inhibited by treatment with an appropriate drug
  24. 24. General Procedure for Performing a RIA Analysis • A known quantity of an antigen is made radioactive • This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. • a sample of serum from a patient containing an unknown quantity of that same antigen is added • This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen for antibody binding sites • As the concentration of "cold" antigen is increase, more of it binds to the antibody. Radio immuno assay 24
  25. 25. General Procedure for Performing a RIA Analysis • And by displacing the radio labelled variant and reduces the ratio of antibody-bound radio labelled antigen to free radio labelled antigen. • The bound antigens are then separated from the unbound ones • the radioactivity of the free antigen remaining in the supernatant is measured. • separating bound from unbound antigen is crucial • Initially, the method of separation employed was the use of a second "anti-antibody" Radio immuno assay 25
  26. 26. Radio immuno assay 26
  27. 27. Advantages Of RIA  Radio immuno assay is very sensitive technique used to measure concentrations of antigen without the need to use a bioassay. It can measure one trillionth (10-12) of a gram of material per milliliter of blood.  It is structurally specific as antigen: antibody reaction are highly specific.  It is indirect method of analysis.  It is a saturation analysis as active reagent added in smaller quantity than that of analyte. Radio immuno assay 27
  28. 28. Disadvantages Of RIA •Prolonged reaction time (in days) as a consequence highly diluted reagent is used. •Radioactive Iodine used in is not a cheap reagent. •Possible health hazards due to handling of radioisotopes. •All the reagents must be added precisely. •Limited assay range. •Lack of direct linear relationship between analyte concentration and signal response. •Difficulty of automation. •Lengthy counting time. Radio immuno assay 28
  29. 29. Uses for RIA  Narcotics (drug) detection,  Blood bank screening for the hepatitis (a highly contagious condition) virus,  Early cancer detection,  Measurement of growth hormone levels,  Tracking of the leukemia virus,  Diagnosis and treatment of peptic ulcers, and  Research with brain chemicals called neurotransmitters Radio immuno assay 29
  30. 30. Radio immuno assay 30 Recent applications…. Refinement of Zalcitabine pharmaco kinetics. Estradiol measurement in translational studies of breast cancer. Plasma estrogen measurement with use of radioimmunoassay has been instrumental in the development of aromatase inhibitors for endocrine therapy of postmenopausal breast cancer Significance of prostate specific antigen in prostate cancer patients and in non cancerous prostatic disease patients. Evaluation of enzyme immunoassay and radioimmunoassay methods for the measurement of plasma oxytocin
  31. 31. Radio immuno assay 31 INSTRUMENTATION • Instrument name: Gamma Counter (WIZARD2) •
  32. 32. Radio immuno assay 32 References Lanning PE. Estradiol measurement in transitional studies of breast cancer. Elsevier 2015;99:26-31. Available from: URL:http://www.elsevier.com/locate/steroids. Muhammed H, Parveen Z, Shahid K, Samreen I, Ajasrasool s. Significance of prostate specific antigen in prostate cancer patients and in non cancerous prostatic disease patients. Journal of Pakistan medical association 2007;57:248-51. John MA, Mark JS, Ross GH, Gene DM, Thaddeus HG et.al. Zalcitabine population pharmacokinetics: application of radioimmunoassay. American society for microbiology 1998;42(2):409-13. Available from: URL:http://aac.asm.org. Angela S, Philip MM, Daniel AN, Benjamin A, Tabak MA et.al. Evaluation of enzyme immunoassay and radioimmunoassay method for the measurement of plasma oxytocin. National institute of health, physchosom med 2011 june;73(5):393-400. SabahA.Presentation on radioimmunoassay. Department of pharmacology. Newdelhi URL:http://www.slideshare.com/radioimmunoassay. Yalow R, Berson S. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest 1960; 39: 1157-75.
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  34. 34. THANK YOU………. Radio immuno assay 34