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
• Originally developed by Yalow and
Berson in 1960 for insulin
Introduction , Theory, Preparation of the Reagents, An actual
Assay , Conclusions

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

Antibody
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Labeled Labeled Antigen

Antigen + Sample
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•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,
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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,
Theory, Preparation
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Tracer, An
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Introduction, Theory,
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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.
Introduction, Theory,
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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
Introduction, Theory,
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Assay, An Actual Assay: Progesterone
Conclusions
Conclusions

• Total count tubes (P4)

• 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
Introduction, Theory,
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of of
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An Actual Assay: Progesterone
Conclusions
Conclusions

(P4)
• Incubate
• Decant
• Count
• Calculate
Introduction,
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An Actual Assay:
Conclusions
Conclusions

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

Std. Curve
[] log[] % mean dec. logit
bindi % % bindin
ng g
0.3 -0.52 79.9 79.9 0.80 1.38
0.6 -0.22 70.2 70.2 0.70 0.86
1.25 0.10 58.5 58.5 0.59 0.34
2.5 0.40 46.6 46.6 0.47 -0.14
5 0.70 34.6 34.6 0.35 -0.64
10 1.00 23.3 23.3 0.23 -1.19

  Coefficien Standard t Stat P-value

ts Error
Intercept 0.504695 0.013009 38.79629 2.64E-06
X Variable -1.67631 0.022652 -74.004 2E-07
1
Introduction,
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An Actual Assay:
Progesterone (P4)
Samples
• Calculate mean % binding for each sample

• Calculate logit % binding for each sample

• Solve: Y = β 0 + β1 X where Y = logit (%binding), X = log

[sample]

• Antilog of X = concentration of antigen in samples

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
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.