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Radioimmunoassay (RIA)

Radioimmunoassay (RIA). Rick McCosh. Introduction , Theory, Preparation of the Reagents, An actual Assay , Conclusions . RIA. Purpose is to determine the concentration of an antigen in solution Competitive binding assay

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Radioimmunoassay (RIA)

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  1. Radioimmunoassay(RIA) Rick McCosh

  2. Introduction , Theory, Preparation of the Reagents, An actual Assay , Conclusions RIA • Purpose is to determine the concentration of an antigen in solution • Competitive binding assay • Originally developed by Yalow and Berson in 1960 for insulin

  3. Introduction , Theory, Preparation of the Reagents, An actual Assay , Conclusions RIA • Reagents • Tracer: labeled antigen • Antibody • Standards: Known concentrations of unlabeled antigen • Unknown samples

  4. Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Antibody

  5. Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Labeled Antigen + Sample Labeled Antigen

  6. Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions • Separate bound from free: • Antibody labeled tubes can be simply decanted • Liquid-phase antibodies need to be precipitated • Use a second antibody • PEG • Centrifugation

  7. Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Count gamma emission • Counts per minute (CPM) for each tube • A sample containing a higher concentration of the unknown antigen will have a lower CPM

  8. Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions

  9. Introduction, Theory,Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Preparation of the Reagents:Antibodies and Antigens • 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.

  10. Introduction, Theory,Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions Preparation of the Reagents:Iodination of the antigen • I125 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. • I125 can be added to the tube and will bind to the oxidized residues • Column chromatography is used to purify the tracer

  11. Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions An Actual Assay: Progesterone (P4) • Total count tubes • Polypropylene tube • Tracer • Non-specific Binding • Polypropylene tube • Tracer • B0 • Antibody labeled tube • Tracer • Standards ( 10, 5, 2.5, 1.25, 0.6125, 0.3125 ng/mL ) • Antibody labeled tube • Tracer • Standard • High and Low pools • Antibody labeled tube • Tracer • High and low pools • Samples containing unknown samples • Antibody labeled tube • Tracer • sample

  12. Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions An Actual Assay: Progesterone (P4) • Incubate • Decant • Count • Calculate

  13. Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions An Actual Assay: Progesterone (P4)Std. Curve • Each tube- Mean NSB = Corrected CPM • Corrected CPM / B0 = % Binding • Logit % binding = Ln(% binding / 1- % binding) • For Standard Curve: • Use SL regression to fit the model: Y = β0 + β1 X where Y = logit (%binding), X = log [sample],

  14. Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions Std. Curve

  15. Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions An Actual Assay: Progesterone (P4)Samples • Calculate mean % binding for each sample • Calculate logit % binding for each sample • Solve: Y = β0 + β1 X where Y = logit (%binding), X = log [sample] • Antilog of X = concentration of antigen in samples

  16. Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions Conclusions: • RIA is an effective, precise and accurate method of quantifying concentrations of an antigen. • Does require approval and training to work with radioactive materials • Modifying an assay procedure can be difficult and time consuming

  17. References Yalow R, Berson S. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest 1960; 39: 1157-1175. Abraham G. Radioimmunoassay of steroids in biological fluids. J. Steroid Biochemistry 1975; 6: 261-270.

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