Immunoassays: ELISA, RIA and Luminex™

by Peter Delves, PhD

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    00:01 ELISA - Enzyme Linked Immunosorbent Assay, is a very, very commonly employed analytical method in immunology.

    00:16 Plastic plates are coated with an antigen.

    00:20 They can be coated with an antibody as well, but in this particular example we are coating the plates with an antigen.

    00:27 Very often one will purchase already coated plates from a manufacturer.

    00:36 The excess antigen is washed off.

    00:41 Test antibody is then added.

    00:44 So maybe this is an autoantigen that is coated on the plate.

    00:48 And we want to discover whether a patient has autoantibodies against that autoantigen.

    00:57 If they do, then they will bind, those autoantibodies will bind to the autoantigen.

    01:04 Following a period of incubation, the plate is washed.

    01:09 And then a ligand to the test antibody is added.

    01:13 In this case the ligand is another antibody coupled to the enzyme horseradish peroxidase.

    01:23 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.

    01:56 Varying amounts of antigen are added.

    02:00 Any unbound antigen is then removed by washing.

    02:06 So for example, maybe we want to measure the amount of a particular substance in the serum of a patient.

    02:17 This will be detected by a specific antibody.

    02:25 Following washing, a labeled second antibody is added that is specific for a non-overlapping epitope of the antigen.

    02:36 So let’s say we are trying to measure a cytokine.

    02:42 The first antibody coating the plate would be for one epitope on that particular cytokine.

    02:49 The other antibody would be for a different epitope, a different part of the same cytokine.

    02:56 Labels include radioactive isotopes or an enzyme that causes a substrate to change color or emit light.

    03:09 This would be in a chemiluminescence assay.

    03:15 The unbound labeled second antibody is removed by washing.

    03:21 And then the amount of second antibody bound is measured.

    03:28 What is able to determine the amount of bound second antibody as a function of the concentration of antigen added with construction of a standard curve.

    03:40 In the fluorescent microsphere Luminex assay, there are beads that are coated with particular antigens.

    03:50 Patient’s serum is added to the coated beads and then incubated.

    03:57 There is a reporter tag, for example fluorescently labeled anti-human IgG antibody added to detect the binding of the patient’s antibody to the antigen coating the bead.

    04:12 These beads are then subject to a technique called flow cytometry, and signals that identify each bead are emitted and detected.

    04:24 There is also a second signal that identifies the reporter tag.

    04:31 The amount of signal is proportional to in this example, anti-hepatitis A antibody in the patient’s serum.

    04:39 So in this particular example, we have beads coated with different antigens from Hepatitis A, B and C and one can detect that the patient has antibodies to Hepatitis A.

    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. Binding of a labeled antibody to the target antigen, which is bound to a monoclonal antibody on a microtiter plate.
    2. Binding of a labeled antibody to 2 antigens of different origins which are coated on to a microtiter plate.
    3. Binding of 2 antigen coated red blood cells to a labeled antibody on a microtiter plate
    4. Precipitating red blood cells using antibodies that specifically bind to their membranes
    5. Isolating an antigen from a solution using an antibody that specifically binds to that particular antigen
    1. Antigen-coated beads
    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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