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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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ELISA:Disadvantages 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 oligomer monomer 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!!
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