1. Radioimmunoassay (RIA)
Rick McCosh

2. 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

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

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

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

6. Separate bound from free:
Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions
Introduction, Theory, Preparation of the Tracer, 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

7. Count gamma emission Counts per minute (CPM) for each tube
Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions
Introduction, Theory, Preparation of the Tracer, 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

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

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

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

11. An Actual Assay: Progesterone (P4)
Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions
Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions
An Actual Assay: Progesterone (P4)
Total count tubes
Polypropylene tube
Tracer
Non-specific Binding B0
Antibody labeled tube
Standards ( 10, 5, 2.5, 1.25, , ng/mL )
Standard
High and Low pools
High and low pools
Samples containing unknown samples
sample

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

13. An Actual Assay: Progesterone (P4) Std. Curve
Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions
Introduction Theory, Preparation of the Tracer, 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],

14. Introduction Theory, Preparation of the Tracer, An actual Assay , Conclusions
Std. Curve []
log[]
% binding
mean %
dec. %
logit binding
0.3
-0.52
79.9
0.80
1.38
0.6
-0.22
70.2
0.70
0.86
1.25
0.10
58.5
0.59
0.34
2.5
0.40
46.6
0.47
-0.14 5
34.6
0.35
-0.64 10
1.00
23.3
0.23
-1.19
Coefficients
Standard Error
t Stat
P-value
Intercept
2.64E-06
X Variable 1
2E-07

15. An Actual Assay: Progesterone (P4) Samples
Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions
Introduction Theory, Preparation of the Tracer, 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

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

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: