1 / 17

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

navid
Download Presentation

Radioimmunoassay (RIA)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.

More Related