Immunoassay

Immunoassay

• 1.
   Radioimmunoassay (RIA)involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity.  The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g.,  Blood banking Diagnosis of allergies Endocrinology  The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. Radioimmunoassay Competitive binding assay. Principle of Radioimmunoassay  Principle: Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ag*  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]
• 2.
  Requirements for RIA 1.Preparation and characterisation of the Antigen [Ligand to be analyzed] 2. Radiolabelling of the Antigen 3. Preparation of the Specific Antibody 4. Development of Assay System • Reagents – Tracer: labeled antigen – Antibody – Standards: Known concentrations of unlabeled antigen – Unknown samples Preparation and Radiolabelling of the Antigen  Antigens prepared by.. – Synthesis of the molecule – Isolation from natural sources  Radiolabelling [Tagging procedure]  Internally Labeled Antigen – 14C and 3H  Externally Labeled Antigen – 131I and 125I Tagging should NOT affect Antigenic specificity and Antigenic activity!
• 3.
  Preparation of theSpecific 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 e.g: Gastrin, Morphine, – Haptens conjugated to albumin  antigenic Separation of Bound and Free Ligand • Electrophoresis • Gel Filtration • Adsorption Chromatography • Fractional Precipitation – Centrifugation – Filtration • Partition Chromatography – Dialysis Assay Procedure  Add known amounts of the test sample + labelled antigen into the microtitre wells  Incubate  allow the reaction to reach completion  Decant and 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 Gamma Counter
• 4.
  • From thesedata, a standard binding curve, like the one shown in red, can be drawn. • The samples to be assayed (the unknowns) are run in parallel. • After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve. Applications of Radioimmunoassays • Endocrinology – Insulin, HCG, Vasopressin – Detects Endocrine Disorders – Physiology of Endocrine Function • Pharmacology – Morphine – Detect Drug Abuse or Drug Poisoning – Study Drug Kinetics Advantages and 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.
• 5.
  Enzyme Linked ImmunosorbentAssay (ELISA)  ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g., a hormone or drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or EIA.  ELISA has many of the advantages (e.g., sensitivity, ease of handling multiple samples) without the disadvantages of dealing with radioactivity (like in RIA).  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 Enzyme labels Assay procedure – 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
• 6.
  • Advantages ofELISA • Sensitive: nanogram levels or lower • Reproducible • Minimal reagents • Qualitative and Quantitative – Qualitative  e.g. HIV testing – quantitative assays  e.g. Hormonal level • Greater scope : Wells can be coated with Antigens OR Antibodies • Suitable for automation high speed • NO radiation hazards