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

9. 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

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

11. 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

12. 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

13. 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

14. 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

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

16. Plasma cell function

17. Antigen localization in Spleen

18. Flow Cytometry

20. DONE!!