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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

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slide2

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
slide3

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

RIA
  • Reagents
    • Tracer: labeled antigen
    • Antibody
    • Standards: Known concentrations of unlabeled antigen
    • Unknown samples
slide4

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

Antibody

slide5

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

slide6

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
slide7

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
slide8

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

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.
preparation of the reagents iodination of the antigen

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
an actual assay progesterone p4

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

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
an actual assay progesterone p4 std curve

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],

std curve

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

Std. Curve
an actual assay progesterone p4 samples

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
conclusions

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
references
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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