Precipitin.
Radioimmunoassay
• Radioimmunoassay (RIA) is a very sensitive in
vitro assay technique used to measure
concentrations of antigens (for example, hormone
levels in the blood) by use of antibodies.
• RIA technique is extremely sensitive and
extremely specific, requiring specialized
equipment.
History
• The technique was introduced in 1960 by
berson and yalow as an assay for the
concentration of insulin in plasma.
• It represented the first time that hormone
levels in the blood could be detected by an in
vitro assay.
• The technique of radioimmunoassay has
revolutionized research and clinical practice in
many areas, which include:
– Blood banking
– Diagnosis of allergies
– Endocrinology
Principle :
• The technique is based on the ability of an unlabelled
form of the substance to inhibit competitively the
binding of a radioactively labelled substance by
specific antibodies.
Method
• To perform a radioimmunoassay, a known
quantity of an antigen is made radioactive,
frequently by labelling it with gamma-
radioactive isotopes of iodine attached to
tyrosine (hot).
• This radiolabelled antigen is then mixed with a
known amount of antibody for that antigen,
and as a result, the two specifically bind to one
another
• Then, a sample of serum from a patient containing an
unknown quantity of that same antigen is added.
• This causes the unlabeled (or "cold") antigen from
the serum to compete with the radiolabelled antigen
("hot") for antibody binding sites.
• As the concentration of "cold" antigen is increased,
more of it binds to the antibody, displacing the
radiolabelled variant, and reducing the ratio of
antibody-bound radiolabelled antigen to free
radiolabelled antigen.
The bound antigens are then separated from the
unbound ones, and the radioactivity of the free
antigen remaining in the supernatant is
measured using a gamma counter. Using
known standards, a binding curve can then be
generated which allows the amount of antigen
in the patient's serum to be derived.
Requirements for the development of
an RIA
1. Pure antigen : for - standards (μg)
2. Tracer : self-made or commercial.
3. Specific, high-affinity antibody : self-made or
commercial.
4. A method to separate bound and free antigen.
5. (Optional) : A system to extract the antigen from the
sample.
principle
• Radioimmunoassay is a technique for measurement of
the concentration of hormones. It is based on a
principle of serial dilutions. One starts with a
combination of radioactively labelled antigen
(corresponding to the hormone to be measured) and
antibody to that hormone. Then, a specific quantity of
unlabeled, or cold antigen is added to the mixture. The
unlabeled antigen competes with the radioactive
antigen for binding to the antibody and displaces a
proportional amount of it. The unbound antigen is
separated away (by centrifugation, for example) and
the amount of radioactivity remaining is measured
(with a Geiger counter).
• This process is continually repeated, using
progressively greater concentrations of unlabeled
antigen, and a line graph demonstrating the
relationship between concentration of the unlabeled
antigen and the radioactivity remaining is constructed.
• This process creates a standard binding curve. The
competitive binding process is then repeated using the
biological sample to be tested and by comparison the
radioactivity resulting with the standard binding curve,
one can deduce the concentration of the hormone in
the sample of interest.
CALIBRATION CURVE
• From these data, a standard
binding curve, like the one
shown in red, can be drawn.
• The samples to be assayed (the
unknowns) are run in parallel.
• After determining the ratio of
bound to free antigen in each
unknown, the antigen
concentrations can be read
directly from the standard
curve.
Advantages
• Radioimmunoassay is widely-used because of
its great sensitivity.
• Using antibodies of high affinity, it is possible
to detect a few pictograms (10−12 g) of antigen
in the tube.
• The greater the specificity of the antiserum, the
greater the specificity of the assay
Disadvantages :
• The main disAdvantages to radioimmunoassay
are
– Use of radioactivity: hazardous
– Expensive equipment (gamma or beta Counter)
– Both 125I or 131I emit gamma radiation that requires
special counting equipment;
– The body concentrates iodine atoms, radioactive
or not in the thyroid gland where they are
incorporated in thyroxine (T4).
