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Radioimmunoassay (RIA) Rick McCosh. RIA Purpose is to determine the concentration of an antigen in solution Competitive binding assay Originally developed.

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Presentation on theme: "Radioimmunoassay (RIA) Rick McCosh. RIA Purpose is to determine the concentration of an antigen in solution Competitive binding assay Originally developed."— Presentation transcript:

1 Radioimmunoassay (RIA) Rick McCosh

2 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 Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

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

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

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

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

7 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 Introduction, Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

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

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

10 Preparation of the Reagents: Iodination of the antigen Introduction, Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions 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 tracer

11 An Actual Assay: Progesterone (P4) Total count tubes Polypropylene tube Tracer Non-specific Binding Polypropylene tube Tracer B 0 Antibody labeled tube Tracer Standards ( 10, 5, 2.5, 1.25, , 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 Introduction, Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

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

13 An Actual Assay: Progesterone (P4) Std. Curve Each tube- Mean NSB = Corrected CPM Corrected CPM / B 0 = % Binding Logit % binding = Ln(% binding / 1- % binding) For Standard Curve: – Use SL regression to fit the model: Y = β 0 + β 1 X where Y = logit (%binding), X = log [sample], Introduction Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

14 Std. Curve []log[] % binding mean %dec. % logit binding Introduction Theory, Preparation of the Tracer, An actual Assay, Conclusions Coefficients Standard Errort StatP-value Intercept E-06 X Variable E-07

15 An Actual Assay: Progesterone (P4) Samples Calculate mean % binding for each sample Calculate logit % binding for each sample Solve: Y = β 0 + β 1 X where Y = logit (%binding), X = log [sample] Antilog of X = concentration of antigen in samples Introduction Theory, Preparation of the Tracer, An actual Assay, ConclusionsIntroduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

16 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 Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions

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


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