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Premium member Presentation Transcript Radioimmunoassay (RIA): Radioimmunoassay (RIA)RIA: RIA to determine the concentration of an antigen in solution involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantization using radioactivity Competitive binding assay Originally developed by Yalow and Berson in 1960 for measurement of insulin in plasmaPrinciple: Principle 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:PowerPoint Presentation: To measure this distribution B-F ,a small but constant amount of labeled antigen ("tracer") is added to the reaction. Eventually, there will be a competition reaction between this small but constant amount of "tracer" and the "cold" antigen for a limited amount of antibody. Since B + F = Ag* = constant, measurement of either B OR F is sufficient.PowerPoint Presentation: 4Ag* + 4 Ab 4Ag*Ab 4Ag + 4Ag* + 4Ab 2Ag*Ab + 2AgAb + 2Ag* + 2 Ab 12Ag + 4Ag* + 4Ab Ag*Ab + 3AgAb + 3Ag* + 9AgRIA: RIA Reagents Tracer: labeled antigen Antibody Standards: Known concentrations of unlabeled antigen Unknown samples (unlabelled antigen= analyte)RIA- The Technique : RIA- The Technique 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. 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, and the radioactivity of each is measured. From these data, a standard binding curve, 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.Standard Curve & Unknown Sample: Standard Curve & Unknown SamplePreparation of the Reagents: : Preparation of the Reagents: Antibodies Polyclonal antibodies are made by injecting an animal with the antigen, then purifying the antibody from serum. Molecules smaller than ~1000 d are not generally immunogenic Steroids are covalently bond to protein carriers which are immunogenic, antibodies can then be purified and their specificity verified. Monoclonal AntibodiesCharacteristics of Tracer: Characteristics of Tracer Must Have Similar Binding Properties as Unlabeled Ligand Internally Labeled Ligand 14 C and 3 H Externally Labeled Ligand 131 I and 125 IPowerPoint Presentation: 1. Internally labeled molecules Typically, tritium ( 3 H) is the label, sometimes 14 C. Usually purchased commercially. Used only for small molecules like steroids or drugs. Merit : the tracer immunologically behaves exactly as the cold hormone, thus theoretically perfect. Demerit : beta-radiation is weak and therefore more difficult to measure, thus practically cumbersome. Beta rays have low penetrating power: the radioactive sample needs to be mixed with a scintillator fluid (flour); the produced light is measured by use of a photomultiplier ("beta-counter").PowerPoint Presentation: 2. Externally labeled molecules Typically, 125 I is the label. Merit : often produced in the research lab itself. gamma-radiation has high penetrating power, is therefore easy to measure, thus practical to use. Demerit : the tracer immunologically does not always behave exactly as the cold hormone, due to iodination damage shelf-life of iodinated protein is < 4 weeks. Usually, 125 I will take the place of a hydrogen atom on on the ring of tyrosine. Sometimes, a radioactively labeled molecule needs to be conjugated to the protein.Preparation of the Reagents: : Preparation of the Reagents: Iodination of the antigen I 125 is the radioactive label most often used. Gamma emission at 35keV Available commercially as NaI Proteins with surface tyrosine groups can be oxidized with commercially available products. I 125 can be added to the tube and will bind to the oxidized residues Column chromatography is used to purify the tracerRIA –Merits & Demerits : RIA –Merits & Demerits versatility : using the same principle, almost any biomolecule can be assayed fast (usually 2 days or less) sensitive (comparable to the most sensitive bioassays, that is < ng/ml) large capacity : thousands of samples/day specific (antibody-dependent) use of radioactivity: hazardous expensive equipment (gamma or beta counter) Does require approval and training to work with radioactive materials Modifying an assay procedure can be difficult and time consumingSeparation of Bound and Free Ligand: Separation of Bound and Free Ligand Antibody labeled tubes can be simply decanted Liquid-phase antibodies need to be precipitated Use a second antibody PEG Electrophoresis Gel Filtration Adsorption Chromatography Partition Chromatography Dialysis Fractional Precipitation Centrifugation FiltrationApplications of Radioimmunoassays: Applications of Radioimmunoassays Endocrinology Insulin, HCG, Vasopressin, T3, T4, TSH, Detects Endocrine Disorders Physiology of Endocrine Function Pharmacology Morphine, digitoxin or digoxin , Detect Drug Abuse or Drug Poisoning Study Drug Kinetics Epidemiology Hepatitis B Clinical Immunology Antibodies for Inhalant Allergens Allergy Diagnosis anti-DNA antibodies Oncology Carcinoembryonic Antigen Early Cancer Detection and DiagnosisPowerPoint Presentation: Gamma Counter Scintillation Counter You do not have the permission to view this presentation. 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