Lab.

#6

(RIA)Radioimmunoassay
(HPLC)High Performance
Liquid Chromatography
1 Miss. Tharaa' Yousef
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.

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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.

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 The technique of radioimmunoassay has revolutionized
research and clinical practice in many areas, which
include:
 Blood banking
 Diagnosis of allergies
 Endocrinology

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: 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.

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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

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 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.

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 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.

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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.

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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.

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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).
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 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.

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 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.

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Advantages

Radioimmunoassay is widely-used because of its great

sensitivity.

Using antibodies of high affinity, it is possible to detect a

few picograms (10−12 g) of antigen in the tube.

The greater the specificity of the antiserum, the greater

the specificity of the assay

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: 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).

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High pressure liquid chromatography (HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

• HPLC is really the automation of traditional liquid

chromatography under conditions which provide for
enhanced separations during shorter periods of time!

• High Performance Liquid Chromatography (HPLC) is one

of the most widely used techniques for identification,
quantification and purification of mixtures of organic
compounds.

• In HPLC, as in all chromatographic methods, components

of a mixture are partitioned between an adsorbent (the
stationary phase) and a solvent (the mobile phase).
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

• The stationary phase is made up of very small

particles contained in a steel column. Due to the small
particle size (3-5 um), pressure is required to force the
mobile phase through the stationary phase.

• There are a wide variety of stationary phases

available for HPLC.

• Silica gel columns are currently one of the most

commonly used HPLC stationary phases.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY