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. 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
oligomer
monomer
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!!