Radioimmunoassay

(RIA)
Rick McCosh
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
Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions
Labeled
Antigen
Labeled Antigen
+ Sample
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

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


Introduction, Theory, Preparation of the Tracer, An actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay , Conclusions
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
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, Preparation of the Reagents, An actual Assay , Conclusions
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
I
125
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.
I
125
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)
Total count tubes
Polypropylene tube
Tracer
Non-specific Binding
Polypropylene tube
Tracer
B
0
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 actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions
An Actual Assay: Progesterone (P4)
Incubate
Decant
Count
Calculate
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 / B
0
= % 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 actual Assay , Conclusions Introduction, Theory, Preparation of the Reagents, An actual Assay, Conclusions
Std. Curve
y = -1.6763x + 0.5047
R = 1
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
2.00
-1.00 -0.50 0.00 0.50 1.00 1.50
L
o
g
i
t

(
%

B
i
n
d
i
n
g
)

log []
P4 Std. Curve
[] log[]
%
binding
mean
% dec. %
logit
binding
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 actual Assay , Conclusions
Coefficients
Standard
Error t Stat P-value
Intercept 0.504695 0.013009 38.79629 2.64E-06
X Variable 1 -1.67631 0.022652 -74.004 2E-07
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 actual Assay , 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

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