Applications of the Immune Response

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Applications of the Immune Response

• Hugh B. Fackrell
• Filename JWR12bv7.ppt

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Content Outline

• Radioimmunoassay (RIA)
• Enzyme Linked Immunosorbent Assay (ELISA)
• Western Blots
• Immunofluorescence
• Immunoelectron Microscopy

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Immunoadsorbent Assays

• Enzyme Linked Immuno Sorbent Assay
• Fluorescent Immuno Sorbent Assay
• Radio Immuno Assay

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Enzyme Linked Immunosorbent Assay (ELISA)

• indirect ELISA
• sandwich ELISA
• Competitive ELISA

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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

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ELISADisadvantages

• Number of separation methods limited
• Expertise required to label and purify conjugates
• Susceptible to interference from non specific
  factors

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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

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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

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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

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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
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Immunofluorescent Methods

• Fluoresecence Immuno Assay
• Fluorescence Quenching
• Fluorescence Enhancement
• Fluorescence Polarization

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Plasma cell function
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Antigen localization in Spleen
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Flow Cytometry
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DONE!!