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RADIOIMMUNOASSAY (RIA)

RADIOIMMUNOASSAY (RIA). Dr. Sigal Fleisher-Berkovich Dept. of Clinical Pharmacology. Principles of the Radioimmunoassay. A fixed concentration of labeled tracer antigen is incubated with a constant amount of antiserum

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RADIOIMMUNOASSAY (RIA)

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  1. RADIOIMMUNOASSAY (RIA) Dr. Sigal Fleisher-Berkovich Dept. of Clinical Pharmacology

  2. Principles of the Radioimmunoassay • A fixed concentration of labeled tracer antigen is incubated with a constant amount of antiserum • If unlabeled antigen is added to the system, there is competition between labeled tracer and unlabeled antigen for the limited number of binding sites on the antibody • The amount of tracer bound to antibody will decrease as the conc. of unlabeled antigen increases • This can be measured after separating antibody bound from free tracer and counting the bound fraction

  3. RIA radioactivity Increasing amount of Insulin

  4. Four basic necessities for RIA • Antiserum to the compound to be measured • 2.A radioactively labeled form of the compound • 3.A method whereby antibody-bound tracer can • be separated from “free” tracer • 4.A standard unlabeled material

  5. Terminology Total Count Tubes: These tubes represent the total amount of radioactivity added in a RIA tube. They have the highest CPM’s (labeled antigen +buffer) Role: quality control to the counts in the rest of the tubes

  6. Terminology Background (blank) tubes: These tubes contain buffer, labeled antigen and charcoal but not antibody They have the lowest CPM’s These counts are considered to be background counts

  7. Terminology Standards: Assay tubes that contain known amounts of the compound to be measured. Those tubes with increasing amount of standard have decreasing CPM’s. RIA curve

  8. Terminology Unknowns: Assay tubes containing the material in which the compound you wish to measure is contained. • Blood serum & plasma • Urine • Saliva • CSF

  9. Terminology Precipitating agent: It is added at the end of incubation It precipitates the “free” labeled and unlabeled antigen Our precipitating agent is dextran-coated charcoal

  10. RIA Table TotalBlankZerostandardassay • No of tubes: 2 2 4 2 2 • buffer (ml) 0.8 0.6 0.1 - - • Standard (ml) - - - 0.1 - • Sample (ml) - - - - 0.1 • Antibody (ml) - - 0.5 0.5 0.5 • Incubation for 30 min • Labeled PG (ml) 0.1 0.1 0.1 0.1 0.1 • Incubation for 1-24 hr • Charcoal0.2 0.2 0.2 0.2

  11. RIA • Advantages: • Extremely sensitive method • Large number of samples can be processed • Small changes in hormone concentrations can be • reproducibly measured • Disadvantages: • Can’t determine if hormone measured has biological • Activity • Peptide hormones can be denatured and not active but still • retain their antigenic character

  12. Prostaglandin E2 Prostaglandins: a 20-carbon unsaturated fatty acids derived from arachidonic acid The inflammatory response is always accompanied by the release of prostaglandins, the predominant product being PGE2 PGE2 mediates: fever, pain vasodilation

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