Immunology

2.  Radioimmunoassays are used to measure the
concentrations of biologically relevant proteins
and hormones in body fluids.
 Although many RIA’s have been replaced by
enzyme based immunoassays, some hormonal
measurements are still made using this
technology.

3. In 1960, two endocrinologists, S A Berson and
Rosalyn Yalow , designed a technique to
determine the level of insulin/ anti insulin
complexes in diabetic patients
Their technique soon proved its value for
measuring hormones, serum proteins , drugs
and vitamins .
Thus RIA’s made it possible to measure
substances that had been undetectable by any
quantitative methodology.

4.  Since the initial description of Yalow’s assay, many
technical variations have been developed to make
the method more rapid and reliable.
 However all these variations depend on the
availability of radioactively labeled antigen or
antibody, and a method by which to separate
antigen-antibody complexes from unbound
reagents.
 Most RIA’s, is used are based on the binding of the
antibody or antigen to a solid phase support, such
as PVC wells of a microtiter plate .
 The radioactive label that most commonly used
is I 125 ,which binds to exposed tyrosine
residues on proteins.

5.  The labeled antigen is mixed with antibody at
a concentration that saturates the antigen
binding site of antibody .
 Then test sample of unlabelled antigen of
unknown concentration are added in
progressively larger amounts .
 The antibody does not distinguish labeled from
unlabeled antigen, so the two kinds of antigen
compete for available binding site on the
antibody .

6.  As the concentration of the unlabeled antigen
increases , more labeled antigen will be displaced
from the binding sites .
 The decrease in the amount of the radio labeled
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.
 The antigen is generally labeled with a gamma –
emitting isotope such as iodine 125 ,but the beta
emitting isotope such as tritum(3H) can also be
used.
 The radio labeled antigen is part of the assay
mixture , the test sample may be complex mixture ,
such as serum or other body fluids that contains
unlabeled antigen.

7.  The procedure requires small amount of sample
and can be conducted in small 96 well microtiter
plates ; hence the procedure is suitable to
determine the concentration of the particular
antigen in large number of samples .
 Eg – a microtiter RIA can be used to screen for the
presence of the hepatitis B virus.
 To determine the amount of labeled antigen bound
, the Ag-Ab complex is precipitated to separate it
from free antigen and the radioactivity in the
precipitate is measured.
 A standard curve can be generated using
unlabeled antigen samples of known concentration
(in place of test sample) and from this plot the
amount of antigen in the test mixture may be
precisely determined.