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Applications of the Immune Response

Applications of the Immune Response. Hugh B. Fackrell Filename JWR12bv7.ppt. Content Outline. Radioimmunoassay (RIA) Enzyme Linked Immunosorbent Assay (ELISA) Western Blots Immunofluorescence Immunoelectron Microscopy. Immunoadsorbent Assays. E nzyme L inked I mmuno S orbent As say

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Applications of the Immune Response

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  1. Applications of the Immune Response Hugh B. Fackrell Filename JWR12bv7.ppt

  2. Content Outline • Radioimmunoassay (RIA) • Enzyme Linked Immunosorbent Assay (ELISA) • Western Blots • Immunofluorescence • Immunoelectron Microscopy

  3. Immunoadsorbent Assays • Enzyme Linked Immuno Sorbent Assay • Fluorescent Immuno Sorbent Assay • Radio Immuno Assay

  4. Enzyme Linked Immunosorbent Assay (ELISA) • indirect ELISA • sandwich ELISA • Competitive ELISA

  5. ELISA: Advantages • Specific & Sensitive- Wide Application • Equipment cheap & available • Reagents “Cheap”, long shelf life • Assays may be rapid • Simultaneous assays; variety of labels • Potential for automation • no radiation hazards

  6. ELISA:Disadvantages • Number of separation methods limited • Expertise required to label and purify conjugates • Susceptible to interference from non specific factors

  7. RIA: Advantages • Measurement simple, not affected by composition of sample matrix • Sensitivity & precision not dependent on the measurement of the magnitude of the signal • Large variety of radiolabelled compounds • labels do not affect reaction kinetics • Mathematically documented

  8. RIA: Disadvantages • Labelled reagents have short shelf life • Potential health hazards • Disposal of radioactive wastes • Equipment is expensive • Variability between batches of labels • Dependence on duration of count time may limit sensitivity of assays

  9. Western Blot • Electrophoresis proteins to separate • Molecular weight, charge, pI etc • 2D electrophoresis possible • Immobilize separated proteins • Electrophoresis onto nitrocellulose • Develop as an ELISA • Product MUST be INSOLUBLE chromogen

  10. oligomer monomer Western Blot with MABS • Same antigen was exposed to 6 different MABS • Staphylococcal alpha toxin • Each MAB reacted with a monomer and a oligomer form of the toxin Maria Sawicki 1996

  11. Immunofluorescent Methods • Fluoresecence Immuno Assay • Fluorescence Quenching • Fluorescence Enhancement • Fluorescence Polarization

  12. Plasma cell function

  13. Antigen localization in Spleen

  14. Flow Cytometry

  15. DONE!!

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