RADIOIMMUNOASSAY

PRESENTED BY:
NAZNEEN SULTANA
 What is RIA??
Radioimmunoassay (RIA) is a very sensitive in-vitro
assay technique used to measure concentrations
of antigens (for example, hormone levels in blood) by use of
antibodies.
RIA technique is extremely sensitive and extremely specific,
and requiring specialized equipment

“Immuno” refers to an immune response that causes the

body to generate antibodies and “assay” refers to a test.

Antigen(Ag)= invading substance for which response produced.

Antibody(Ab)= protein produce in body in response to antigen.
 Principle
The basic principle of radioimmunoassay is
competitive binding, where a radiolabeled or
radioactive antigens ("tracer") compete with a non-
radioactive or unlabeled antigens for a fixed, limited
number of binding sites on the specific antibody or
receptor binding sites.
Ag*+nAb nAg*Ab
+ Ag
AgAb+ Ag*Ab + Ag + Ag*
unlabeled labeled unbound antigen
complex complex
 Cont….
O Radioimmunoassay (RIAs) utilize a radioactive
label(usually 125I, 3H or 14C), which emits radiation that
can be measured with a beta or gamma counter.
O The amount of Ab is kept constant, the amount of
antigen added (known or unknown) is variable.
O The added antigen will be distributed between a
bound (B) and a free (F) fraction.
O Increasing amounts of unlabeled antigen(Ag) in the
sample will compete with tracer(Ag*) for antibody(Ab)
for binding, leading to more unlabeled antigen-
antibody complex (Ag-Ab)formation.
 Method
Known quantity of an antigen is made
radioactive by labeling it with gamma-
radioactive isotopes(eg. 125 I).

Radiolabeled antigen is then mixed with

a known amount of antibody for that
antigen.

Antigen-antibody complex
formed(leveled).

Known quantity of unlabeled antigen is

added to mixture.

Unlabeled antigen compete with

radiolabeled antigen for antibody binding
sites.
 As the concentration of unlabeled
antigen is increased, more of it
binds to the antibody, displacing the
radiolabeled variant.

The ratio of antibody-bound

radiolabeled antigen to free
radiolabeled antigen reduces.

The bounded antigen is then

separated from the unbound ones.

The radioactivity of free antigen

remaining in supernatant and bound
in precipitate is measured using
radioactive counter.

From the data obtained, a standard

binding curve is plotted with ratio of
bound to free antigen on Y-axis and
unlabeled antigen on X-axis.
The samples to be assayed (the unknowns) are run
in parallel.

After determining the ratio of bound to free

antigen ("cpm Bound/cpm Free") in each
unknown, the antigen concentrations can be
read directly from the standard curve
Instrumentation for RIA
O Centrifuge: For separating the bound
radiolabelled antigens from the unbound
radiolabelled antigens.
O Radioactive counters:
O Liquid Scintillation counters: Radioactive
isotopes that emit β-radiations are used (Eg:
3 H, 14 C).

O Gamma counters: Radioactive isotopes that

emit γ-radiations are used(Eg: 124-I, 125-I,
131-I).
RADIOACTIVE COUNTER
O RADIOACTIVITY is process by which a
unstable nuclei(radioactive ) loses energy by
emitting alpha, beta or gamma rays.
O The SI unit of radioactive activity is the
becquerel (Bq), named in honour of the
scientist Henri Becquerel.
O Older unit is curie(Ci).
1 curie (Ci) = 3.7×1010 Bq
Nt=Noe(-0.693t/t0.5)
 Gamma counters
O Sample is placed in sealed vials or test tubes, and
moved along a track.
O It move inside a shielded detector.
O Within this shielded detector there is a scintillation
crystal that surrounds the radioactive sample.
O Gamma rays emitted from the radioactive sample
interact with the crystal, are absorbed, and light is
emitted.
O A detector, such as a photomultiplier tube converts the
visible light to an electrical signal.
O Electrical signal is measured by spectrometer.
O Depending on the half-life and concentration of the
sample, measurement times may vary from 0.02
minutes to several hours.
Gamma counter(WIZARD2)
 Liquid scintillation counting
O The compound containing small amount of radionuclide
is dissolved in a liquid scintillation.
O Tracer labels the sample and allows the molecule to be
traced.
O Emitted beta particles collide with solvent molecule
leading to release of energy.
O This energy is absorb by scintillator compound which is
emitted in the form of fluorescence.
O This is detected by photomultiplier tube and converted
into electrical energy for counting by spectrometer.
 Application
 Narcotics (drug) detection.
 Blood bank screening for the hepatitis virus.
 Early cancer detection.
 Measurement of growth hormone levels.
 Tracking of the leukaemia virus.
 Diagnosis and treatment of peptic ulcers.
 Research with neurotransmitters.
 Detecting infection.
 Detecting allergens in food and house dust.
 Measurement of plasma oxytocin.
 Estradiol measurement in studies of breast cancer.