A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.

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RIA
(Radioimmunoassay)
Introduction, Principle and Applications
INTRODUCTION:
 A radioimmunoassay (RIA) is an immunoassay that uses radiolabeled molecules in a stepwise
formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure
concentrations of substances, usually measuring antigen concentrations (for
example, hormone levels in blood) by use of antibodies.
 Although the RIA technique is extremely sensitive and extremely specific, requiring specialized
equipment, it remains among the least expensive methods to perform such measurements.
The Technique:
 A mixture is prepared of
o radioactive antigen
 Because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein,
the radioactive isotopes 125
I or 131
I are often used.
o Antibodies ("First" antibody) against that antigen.
 Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for
the binding sites of the antibodies.
 At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced
from the antibody molecules.
 The antibody-bound antigen is separated (see below) from the free antigen in the supernatant fluid, and the
radioactivity of each is measured.

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Note: Please, for more details refer books
PRINCIPLE:
Principle of Radioimmunoassay (RIA)
 Radioimmunoassay uses radioisotope-labeled purified known antigen which competes with
unlabeled (unknown) antigen for binding sites on a known amount of antibody.
 The Ag-Ab complexes that form between the antigen and antibody can then be precipitated using the
second antibody and the amount of radioactivity of the bound complex is measured by means of
radioisotope analyzers and autoradiography.
 When the sample contains a high amount of antigen, much of the antigen-binding sites of the antibodies
are occupied by unlabeled antigen. So the bound complex will show little radioactivity. The
radioactivity generated, in fact, is inversely proportional to the amount of antigen present in the sample.
 Then the concentration of the unknown (unlabeled) antigen or hapten present in a sample is determined
by plotting the value of radioactivity generated in a standard chart generated using different
concentrations of same antigens against the radioactivity of the bound complex.
APPLICATIONS OF RIA:
 The test can be used to determine very small quantities (e.g. nanogram) of antigens and antibodies in
the serum.
 The test is used for quantitation of hormones, drugs, HBsAg, and other viral antigens.
 Analyze nanomolar and picomolar concentrations of hormones in biological fluids.
LIMITATIONS
 The handling of radioisotopes requires specific safety measures. Because of this limitation, RIA has
been replaced by ELISA in clinical laboratories.