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Radioimmunoassay
Immunoassay reactions may be competitive or non-competitive
 Competitive
labeled known and patient unknown are added to reaction and “compete” for
the target.
 Non-competitive
– Add patient sample, for example looking for antibody, to known reagent
antigen.
– Reaction occurs and the concentration is directly related to the amount
of antibody in patient sample.
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Radioimmunoassay (RIA) is a very sensitive Competitive
binding assay used to measure concentrations of antigens (for
example, hormone levels in blood) using the specificity of antigen
– antibody binding and quantitation using radioactivity.
Radioactive isotopes are usually high specific activity H3 (beta) ,
I125 (gamma).
This technique used for detection of micro-quantites of proteins,
hormones, viral antigens, antibodies, vitamins and drugs.
RIA is used in various branches of science like Biochemistry,
microbiology, Hematology, endocrinology and Clinical
Pharmacology.
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Highly specific (antibody dependent)
One of the most sensitive techniques for detecting
antigen or antibody, it is possible to detect a few
picograms (10^-12 g) of hormone in the tube.
 Easy signal detection
 Well-established, rapid assays
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1. Hazardous due to use of radioactivity
2. The time and expense associated with maintaining a licensed
radiation safety and disposal program.
3. The health and safety risks posed by the use of radiation
4. Need expensive equipments as γ or β counter & Radiolabeled
Isotopes
5. The body concentrates iodine atoms — radioactive or not — in
the thyroid gland where they are incorporated in thyroxine (T4)
synthesis result in hypothyroidism
For these reasons, RIA has largely replaced in routine clinical laboratory by
enzyme immunoassay.
It requires special precautions and licensing,
since radioactive substances are used.
1. Pregnant females should not work in an area
where RIA tests are being performed.
2. Personnel handling isotope reagents must
wear lead-coated gown & gloves to limit their
exposure to radiation.
3. Special sinks and waste disposal containers
are required for disposal of radioactive waste.
4. The amount of radioisotope discarded must be
documented for both liquid and solid waste.
5. Leakage or spills of radioactive reagents must be
measured for radioactivity; the amount of
radiation and containment and disposal processes
must be documented.
6. The work must be in a lab with lead-covered floor or
another radioabsorbent leather.
7. Radioisotope waste should be disposed in a special
way.
Based on competition between unlabelled antigen and
finite amount of corresponding labeled antigen usually
with Iodine 125 for a limited number of high-affinity
antibody binding sites in a fixed amount of antiserum.
 The antibody does not distinguish labeled from
unlabeled antigen, so the two kinds of antigen compete
for available binding sites on the antibody.
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As the concentration of unlabeled antigen increases, more
labeled antigen will be displaced from the binding sites. The
amount of radiolabeled antigen bound to specific antibody in
the presence of the test sample is measured in order to
determine the amount of antigen present in the test sample.
Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ag*
Relationship between radiolabeled Ag and unlabeled (serum’s) Ag
. Ab-coated plastic tubes in addition to
Total count tube
Non-specific Binding tube
2. Calibrators with known cons. of
unlabeled Ag
3. Sample Diluents
4. I125 ﴾radio-labeled Ag﴿
5. Washing buffer
1
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 Mark all the tubes
Total count
tube
Non-specific
binding tube
Calibrators
0
5
10
15
20
25
Sample/s
Contain labeled Ag only
 Addition of
samples and calibrators ( unlabeled Ag )to their
corresponding tubes.
Addition of labeled Ag ( I125 ) to all the previous tubes
Incubation at room temperature.
Centrifugation for 15 min. at 1500 rpm.
Decant & wash contents of the tubes except Total tube
The radioactivity of each is measured by Gamma counter
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
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γ-counter
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From these data, a standard
binding curve, like the one
shown in red, can be drawn.
CPM count expresses the
labeled Ag (I125) conc.
As seen in the diagram,
radioactive signal of I125 is
inversely proportional to
unlabeled Ag conc. in sample.
Total binding tube act as a control, neither washed nor
decanted.
 Non-specific binding tube (NSB) contain only labeled Ag I125 ,
used to exclude any residual free iodine after decant step.
 Zero calibrator tube shows maximum binding, there is no Ag
to compete with the labeled Ag for Ab binding sites.
 The competition increases as the concentration of serum Ag
increases, and so I125 binding decreases
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As CPM are high numbers, We can construct another curve
between Ag concentration and percentage of CPM of each tube
using this formula :
%S = CPM S * 100%
CPM MB
S= sample
MB = maximum binding tube
percentage
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Unlabeled Ag conc.
An example of A drug determination by RIA immunoassay
Immunoradiometric assay
(Non competitive)
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The IRMA is a noncompetitive assay in which the analyte to be
measured is 'sandwiched' between two antibodies.
The first antibody is immobilized inside the walls of the
tubes.
The other antibody is radiolabeled for detection.
The analyte present in the samples, standards and controls is
bound by both antibodies to form a 'sandwich' complex.
Advantages of IRMA include a faster reaction rate and an
increased sensitivity because the antibody excess allows all of
the unknown analyte to be involved in the reaction.
It resemble the principle of non-competitive Sandwich ELISA but the label
here is radioactive I125 rather than enzyme and the method of signal
detection certainly different .
Although immunoassays are both highly sensitive and specific, false
positive and negative results may occur.
 False-negative results from
1. improper sample storage or treatment
2. reagent deterioration
3. improper washing technique.
 False-positive results
1. For samples containing small fibrin strands that adhere to the solid phase
matrix.
2. By substances in the blood or urine that cross-react or bind to the antibody
used in the test.