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Radioimmunoassay (RIA) PowerPoint PPT Presentation


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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 - PowerPoint PPT Presentation

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Radioimmunoassay (RIA)

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Radioimmunoassay ria l.jpg

Radioimmunoassay(RIA)

Rick McCosh


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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


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

RIA

  • Reagents

    • Tracer: labeled antigen

    • Antibody

    • Standards: Known concentrations of unlabeled antigen

    • Unknown samples


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

Antibody


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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


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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


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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


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Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions

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


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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.


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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


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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


  • An actual assay progesterone p412 l.jpg

    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


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    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],


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    Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions

    Std. Curve


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    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


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    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


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    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.