RIA

RIA

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  RADIO IMMUNO ASSAY FIRDOUS M.Pharmacy
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  CONTENTS  Introduction  Theory Principle  Requirements  Procedure  Applications
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  INTRODUCTION • Radio immunoassay is an immunological assay to analyse antigens present in given biological samples • It is most sensitive and specific method of immuno assay • 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.
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  THEORY • RIA involvesthe 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 level.
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  PRINCIPLE • It involvesthree 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).
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  REQUIREMENTS • Micro titerplate / Test tubes. • Pure antigen • Radio labelled antigen • Antibodies • Standard's • Centrifuge • Radioactive counter
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  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 liquid.
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  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 half lifes.
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  ANTIBODY • Specific antibodiesare obtained by injecting the Ag to animals. • Ag I.e., drug molecule + bovineserum albumin
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  STANDARDS Un labeled Agconcentration B/F Ratio
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  Centrifuge: • Used foethe separation of precipitated form and supernatant liquid form. • Range: 1200 – 2500 rpm
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  Radio active counters: Two types of counters are used They are : a) Gamma counter. b) Scintillation counters.
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  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
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  PROCEDURE :
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  Test tube-1 Blank Ab +Ag* Testtube -2 Calibrator Ab + pure Ag +Ag* Test tube -3 Sample Ab + sample Ag + Ag* PROCEDURE :
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  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.
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  Applications : Radio immunoassay of morphine : This involves : • Synthesis of immunogen • Antiserum production. • Procedure.
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  CONCLUSION : • RIAis 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.
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  REFERENCE :