Transcript Radioimmunoassay (RIA) - Department of Chemistry and Biochemistry

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,
Antibody
An actual Assay ,
Conclusions
Introduction,
Theory,
Theory,
Preparation
Preparationofofthe
theReagents,
Tracer,
Labeled
Antigen
An actual Assay ,
Labeled Antigen
+ Sample
Conclusions
Introduction,
Theory,
Theory,
Preparation
Preparationofofthe
theReagents,
Tracer,
An actual Assay ,
•Separate bound from free:
•Antibody labeled tubes can be simply decanted
•Liquid-phase antibodies need to be precipitated
•Use a second antibody
•PEG
•Centrifugation
Conclusions
Introduction,
Theory,
Theory,
Preparation
Preparationofofthe
theReagents,
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
Introduction,
Theory,
Theory,
Preparation
Preparationofofthe
theReagents,
Tracer,
An actual Assay ,
Conclusions
Introduction,
Theory,
Theory,
Preparation
Preparationofofthe
theReagents,
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.
Introduction,
Theory,
Theory,
Preparation
Preparationofofthe
theReagents,
Tracer,
An actual Assay ,
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
Conclusions
Introduction,
Theory,
Theory,
Preparation
Preparation
of of
thethe
Reagents,
Tracer,
An
Anactual
actualAssay,
Assay ,
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
Conclusions
Conclusions
Introduction,
Theory,
Theory,
Preparation
Preparation
of of
thethe
Reagents,
Tracer,
An
Anactual
actualAssay,
Assay ,
An Actual Assay: Progesterone (P4)
•
•
•
•
Incubate
Decant
Count
Calculate
Conclusions
Conclusions
Introduction
Introduction,
Theory,
Theory,
Preparation
Preparation
of of
thethe
Reagents,
Tracer,
An
Anactual
actualAssay,
Assay ,
Conclusions
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 = β 0 + β1 X
where Y = logit (%binding), X = log [sample],
Introduction
Theory,
Preparation of the Tracer,
An actual Assay ,
Conclusions
Std. Curve
0.3
0.6
1.25
2.5
-0.52
-0.22
0.10
0.40
79.9
70.2
58.5
46.6
79.9
70.2
58.5
46.6
0.80
0.70
0.59
0.47
1.50
1.38
0.86
0.34
-0.14
5
0.70
34.6
34.6
0.35
-0.64
10
1.00
23.3
23.3
0.23
-1.19
Standard
Coefficients
Error
Intercept
0.504695
0.013009
X Variable 1
-1.67631
0.022652
P4 Std.
Curve
2.00
Logit (% Binding)
[]
%
mean
logit
log[] binding % dec. % binding
-1.00
1.00
y = -1.6763x + 0.5047
R² = 1
0.50
0.00
-0.50
0.00
-0.50
-1.00
-1.50
log []
t Stat
38.79629
-74.004
P-value
2.64E-06
2E-07
0.50
1.00
1.50
Introduction
Introduction,
Theory,
Theory,
Preparation
Preparation
of of
thethe
Reagents,
Tracer,
An
Anactual
actualAssay,
Assay ,
Conclusions
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
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.