Immunoassays: ELISA, RIA and Luminex™

by Peter Delves, PhD

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    ELISA - Enzyme Linked Immunosorbent Assay, is a very, very commonly employed analytical method in immunology. Plastic plates are coated with an antigen. They can be coated with an antibody as well, but in this particular example we are coating the plates with an antigen. Very often one will purchase already coated plates from a manufacturer. The excess antigen is washed off. Test antibody is then added. So maybe this is an autoantigen that is coated on the plate. And we want to discover whether a patient has autoantibodies against that autoantigen. If they do, then they will bind, those autoantibodies will bind to the autoantigen. Following a period of incubation, the plate is washed. And then a ligand to the test antibody is added. In this case the ligand is another antibody coupled to the enzyme horseradish peroxidase. The plate is again washed and a substrate, a chromagen is added and a colored product is produced by the enzyme reaction. The amount of color is proportional to the amount of antibody bound to the antigen. In a sandwich immunoassay, first of all antibody is bound to the well of a microtitre plate, which is typically a 96 well plate. Varying amounts of antigen are added. Any unbound antigen is then removed by washing. So for example, maybe we want to measure the amount of a particular substance in the serum of a patient. This will be detected by a specific antibody. Following washing, a labeled second antibody is added that is specific for a non-overlapping epitope of the antigen. So let’s say we are trying to measure a cytokine. The first antibody coating the plate would be for one epitope on that particular cytokine. The other antibody would be for a different epitope, a different part of the same...

    About the Lecture

    The lecture Immunoassays: ELISA, RIA and Luminex™ by Peter Delves, PhD is from the course Immunodiagnostics. It contains the following chapters:

    • ELISA
    • Sandwich Immunoassay
    • Fluorescent Microsphere (Luminex™) Assay

    Included Quiz Questions

    1. Horseradish peroxidase
    2. Lysozyme
    3. Caspase 3
    4. Activated-induced cytidine deaminase
    5. : C3 convertase
    1. A labeled antibody and an antibody bound to a micotiter plate that can both bind to the same antigen at different regions
    2. An antigen specific antibody with a label that competes for binding with other antibodies
    3. Antibody bound to a ligand specific for the antigen
    4. A label bound to a ligand that recognizes the antibody bound to the microtiter plate
    5. Unbound antibody that can recognize antigen bound to a label
    1. Fluorescent anti-human IgA antibody
    2. Horseradish peroxidase
    3. A radioactive isotope
    4. Chromagen
    5. Caspase 3

    Author of lecture Immunoassays: ELISA, RIA and Luminex™

     Peter Delves, PhD

    Peter Delves, PhD

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