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Radioimmunoassay & Enzyme Linked Immunosorbent Assay M K Unnikrishnan [Aug 2006]

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Principle of Radioimmunoassay Principle: Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab* Unbound Ag* and Ag washed out Radioactivity of bound residue measured Ligand conc is inversely related to radioactivity [Ag : ligand to be measured ; Ag* radiolabelled ligand]

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Advantages & Disadvantages of RIA Advantages Highly specific: Immune reactions are specific High sensitivity : Immune reactions are sensitive Disadvantages Radiation hazards: Uses radiolabelled reagents Requires specially trained persons Labs require special license to handle radioactive material Requires special arrangements for Requisition, storage of radioactive material radioactive waste disposal.

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Requirements for RIA Preparation & characterisation of the Antigen [Ligand to be analysed] Radiolabelling of the Antigen Preparation of the Specific Antibody Development of Assay System

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Preparation & Radiolabelling of the Antigen Antigens prepared by.. Synthesis of the molecule Isolation from natural sources Radiolabelling [Tagging procedure] 3 H 14 C 125 I are used as radioactive tags Antigens are tagged to 3 H 14 C 125 Tagging should NOT affect Antigenic specificity & Antigenic activity !

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Preparation of the Specific Antibody Antigen injected intradermally into rabbits or guinea pigs  antibody production Antibodies recovered from the serum Some ligands are not Antigenic Hormones, Steroids, Drugs  HAPTENS Eg: Gastrin, Morphine, Haptens conjugated to albumin  antigenic

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Development of the Assay System A crucial step is separation of unbound antigens This achieved by binding the antibodies to the microtitre well surface [Solid phase RIA] Antigens bound to the fixed antibodies remain stuck to the inner surface Decanting & washing the well removes unbound antigens Other techniques of separation: Centrifugation

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Assay Procedure Add known amounts of the test sample + labelled antigen into the microtitre wells Incubate  allow the reaction to reach completion Decant & wash contents of the well  removes all unbound antigens Radioactivity remaining in the Microtitre wells measured by a Counter [GM counter , Scintillation counter etc] Intensity of radioactivity is inversely correlated with the conc of antigens in the test sample Sensitive to very low conc of antigens

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Enzyme Linked Immunosorbent Assay Principle: Uses an immune reaction like RIA Differs from RIA in detection method Detection based on Enzyme catalysed reaction OR Fluorescent probe NOT radioactivity [great advantage!]

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Advantages of ELISA Sensitive: nanogram levels or lower Reproducible Minimal reagents Qualitative & Quantitative Qualitative  Eg HIV testing quantitative assays  Eg Ther. Drug Monitoring Greater scope : Wells can be coated with Antigens OR Antibodies Suitable for automation high speed NO radiation hazards

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Types of ELISA Noncompetitive binding assay or Sandwich method Antigen measuring system [Titrewells coated with antibodies ; Enzyme labelled antibodies] Antibody measuring system [Titrewells coated with antigens ; Enzyme labelled antiantibodies] Competitive binding assay [Titrewells coated with antibodies ; Enzyme labelled antigens]

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Noncompetitive or Sandwich Assay Antigen measuring system Titre wells coated with suitable antibody Add patient sample containing the antigen Incubate: till antigen antibody reaction is complete Wash remove unbound antigen Add Antibody labelled with Enzyme Incubate till antigen binds labelled antibody Wash  remove unbound labelled antibody Add substrate ; incubate Enzyme + Substrate  Product  measure colour Colour proportional to antigen in patient sample

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Noncompetitive or Sandwich Assay Antibody measuring system Titre wells coated with suitable antigen Add patient sample containing the antibody Incubate: till antigen antibody reaction is complete Wash remove unbound antibody Add Antiantibody labelled with Enzyme Incubate till labelled antiantibodies binds antigen-antibody complex Wash  remove unbound labelled antiantibody Add substrate ; incubate Enzyme + Substrate  Product  measure colour Colour proportional to antibody in patient sample

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Competitive binding assay Titrewells coated with antibodies Known quantities of patient sample containing antigen + antigen labelled with enzyme Incubate: till antigen antibody reaction is complete Wash remove unbound antigens Add substrate ; incubate Enzyme + Substrate  Product  measure colour Colour inversely related to antigen in patient sample

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Enzyme labels Enzyme labels should have high specific reactivity Should be easily coupled to ligands & the labelled complex must be stable The reactivity should be retained after linking of the enzyme to the antigen/antibody The chosen enzymes should not be normally present in the patient samples Examples of enzyme labels Horse radish peroxidase, Alkaline phosphatase, Glucose oxidase

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Applications of Immunoassays [RIA & ELISA] Analysis of hormones, vitamins, metabolites, diagnostic markers Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids, Therapeutic drug monitoring: Barbiturates, morphine, digoxin, Diagnostic procedures for detecting infection HIV, Hepatitis A, B etc