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• Radio immuno assay is an immunological assay to
analyse antigens present in given biological samples
• It is most sensitive and specific method of immuno
• Sensitivity ranges from 0.0006 -0.006 µg / ml .
• It was developed by S.A. Berson and Rosalyn yalow
in 1959 in received nobel prize in 1977.
• RIA involves the reaction between antigen (hapten) and its specific
Antibody in presence of radio labelled antigen.
• This hapten is obtained from a non-antigenic compound e.g.,
morphine , Cartelol etc., which is ultimately conjugated .
• Animals normally develop antibodies to the injected immunogenic
substances as part of their natural immune response.
• The serum derived from this animals is used as the antibody source
and tested with reference to their specificity, affinity at their titre
• It involves three principles which make it most specific & sensitive than
other immune assays.
• An immune reaction i.e. antigen, antibody binding.
• A competitive binding or competitive displacement
reaction (It gives specificity).
• Measurement of radio emission (It gives sensitivity).
• Micro titer plate / Test tubes.
• Pure antigen
• Radio labelled antigen
• Radioactive counter
Micro titer plate :
• A microtiter plate is used mostly used for this assay
• A microtiter plate could have 6, 24, 96, 384 or even sometimes
1536 wells arranged in rows.
• Each well of a microplate can only hold very small amounts of
PURE ANTIGEN :
• Antigen may be obtained from biological sample or by synthetic form , it
should be pure.
• It is used as standard or calibrator.
RADIO LABELLING OF ANTIGEN:
•The most commonly used radiolabels in RIA are tritium and iodine
• They have adequate activity and have long enough
• Specific antibodies are obtained by injecting the
Ag to animals.
• Ag I.e., drug molecule + bovineserum albumin
Un labeled Ag concentration
• Used foe the separation of
precipitated form and
supernatant liquid form.
• Range: 1200 – 2500 rpm
Radio active counters :
Two types of counters are used
They are :
a) Gamma counter.
b) Scintillation counters.
a)Gamma counter :
• These are used for the gamma energy emitting isotopes
e.g., common iodine isotopes.
b) Scintillation counter :
• These are used for counting beta energy emitting isotopes
e.g., tritium , carbon -14 isotopes
Test tube -2
Ab + pure Ag +Ag*
Test tube -3
Ab + sample Ag + Ag*
SEPARATION TECHNIQUES :
• After completion of reaction of reaction free form and bound forms
are determined by separation techniques.
• Various techniques include gel filtration, Electrophoresis, solid
phase adsorption of Ag, Ab & fractional precipitate.
Radio immuno assay of morphine :
This involves :
• Synthesis of immunogen
• Antiserum production.
• RIA is invitro assay technique used for the determination of Ag concentration
present in given sample.
• In advance to RIA many other techniques are introduced like RAST, EIA , RIST,
lRMA , competitive RIA.