radioimmunoassay (2)

8/7/2019 Radioimmunoassay (2)

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Radioimmunoassay(RIA)

Rick McCosh


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RIA

Purpose is to determine theconcentration of an antigen insolution

Competitive binding assay

Originally developed by Yalow andBerson in 1960 for insulin

Introduction , Theory, Preparation of the Reagents, An actualAssay , Conclusions


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RIA

ReagentsTracer: labeled antigen

Antibody

Standards: Known concentrations of unlabeledantigen

Unknown samples

Introduction , Theory, Preparation of the Reagents, An actualAssay , Conclusions


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Antibody

Introduction, Theory, Preparation of the Reagents, An actualAssay , Conclusions


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Labeled

Antigen

Labeled Antigen

+ Sample

Introduction, Theory, Preparation of the Tracer, An actualAssay , Conclusions



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Separate bound from free:

Antibody labeled tubes can be simplydecanted

Liquid-phase antibodies need to beprecipitated

Use a second antibodyPEGCentrifugation

Introduction, Theory, Preparation of the Tracer, An actualAssay , Conclusions



7/17

Count gamma emission

Counts per minute (CPM) for each tube

A sample containing a higherconcentration of the unknown antigenwill have a lower CPM

Introduction, Theory, Preparation of the Tracer, An actualAssay , ConclusionsIntroduction, Theory, Preparation of the Reagents, An actualAssay , Conclusions


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Introduction, Theory, Preparation of the Tracer, An actualAssay , ConclusionsIntroduction, Theory, Preparation of the Reagents, An actualAssay , Conclusions


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Preparation of the Reagents:Antibodies and Antigens

Introduction, Theory, Preparation of the Tracer, Anactual Assay , Conclusions

Polyclonal antibodies are made by injecting an animalwith the antigen, then purifying the antibody fromserum.

Molecules smaller than ~1000 d are not generallyimmunogenic

Steroids are covalently bond to protein carrierswhich are immunogenic, antibodies can then be

purified and their specificity verified.



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Preparation of the Reagents:Iodination of the antigen

Introduction, Theory, Preparation of the Tracer, Anactual Assay , ConclusionsIntroduction, Theory, Preparation of the Reagents, An actualAssay , Conclusions

I125 is the radioactive label most often used. Gamma emission at 35keV Available commercially as NaI

Proteins with surface tyrosine groups can beoxidized with commercially available products.

I125 can be added to the tube and will bind to theoxidized residues

Column chromatography is used to purify the

tracer


11/17

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

Introduction, Theory, Preparation of the Tracer, An actualAssay , ConclusionsIntroduction, Theory, Preparation of the Reagents, An actualAssay, Conclusions


12/17

An Actual Assay: Progesterone(P4)

Incubate

Decant

Count

Calculate

Introduction, Theory, Preparation of the Tracer, An actualAssay , ConclusionsIntroduction, Theory, Preparation of the Reagents, An actualAssay, Conclusions


13/17

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 = 0 + 1 X where Y = logit (%binding), X = log [sample],

Introduction Theory, Preparation of the Tracer, An actualAssay , ConclusionsIntroduction, Theory, Preparation of the Reagents, An actualAssay, Conclusions


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Std. Curve

[] log[] %binding

mean% dec.% logitbinding

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

Introduction Theory, Preparation of the Tracer, An actualAssay , Conclusions

Coefficien

ts

Standard

Error

t Stat P-value

Intercept 0.504695 0.013009 38.79629 2.64E-06

X Variable1

-1.67631 0.022652 -74.004 2E-07

d i h i f h l

d i h i f h l


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An Actual Assay:Progesterone (P4)

Samples Calculate mean % binding for each sample

Calculate logit % binding for each sample

Solve: Y = 0 + 1 X where Y = logit (%binding), X = log[sample]

Antilog of X = concentration of antigen in samples

Introduction Theory, Preparation of the Tracer, An actualAssay , ConclusionsIntroduction, Theory, Preparation of the Reagents, An actualAssay, Conclusions

I d i Th P i f h R A l


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Conclusions:

RIA is an effective, precise and accurate methodof quantifying concentrations of an antigen.

Does require approval and training to work withradioactive materials

Modifying an assay procedure can be difficult and

time consuming

Introduction, Theory, Preparation of the Reagents, An actualAssay, Conclusions


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References

Yalow R, Berson S. Immunoassay of endogenous plasmainsulin in man. J. Clin. Invest 1960; 39: 1157-1175.

Abraham G. Radioimmunoassay of steroids in biological fluids.

J. Steroid Biochemistry 1975; 6: 261-270.

radioimmunoassay (2)

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