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

Enzyme Linked Immunosorbent Assay (ELISA) indirect ELISA sandwich ELISA Competitive ELISA

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

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

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

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

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

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

Immunofluorescent Methods Fluoresecence Immuno Assay Fluorescence Quenching Fluorescence Enhancement Fluorescence Polarization

Plasma cell function

Antigen localization in Spleen

Flow Cytometry

DONE!!