Immunodiagnostic Methods: Agglutination, Nephelometry, Immunoprecipitation and Radial Immunodiffusion

by Peter Delves, PhD

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    00:01 Agglutination and precipitation can be used for the detection of antibodies or antigens.

    00:10 In the agglutination of antigen-coated particles, there is detection of specific antibodies.

    00:19 In nephelometry, there can be quantification of proteins in serum, plasma or cerebrospinal fluid.

    00:28 Immunoprecipitation can be used for the isolation of specific antigen from a mixture of antigens.

    00:36 And radial immunodiffusion used for the quantification of antigens.

    00:41 In agglutination assays, particles are coated with either antibody or antigen.

    00:48 In this case, the particle is being coated with the IgG antibody.

    00:55 You now have a particle that is sensitized with IgG.

    01:00 A rheumatoid factor negative serum will not cause agglutination of these particles.

    01:09 However if a patient has rheumatoid factor which are autoantibodies directed to the Fc part of IgG, then addition of serum from that rheumatoid factor positive patient will cause agglutination.

    01:27 In nephelometry, the presence of an analyte in a test sample can be detected.

    01:33 Specific polyclonal antibodies are mixed with the sample containing the suspected analyte.

    01:41 These are added to a cuvette and immune complexes will form if there is the analyte for that particular antibody present in the test sample.

    01:54 Laser light is then shone onto the cuvette and there will be light scatter depending upon the amount of immunoprecipitate that is formed.

    02:07 The light scatter is detected using a photodiode.

    02:13 The light scatter is proportional to the number of immune complexes, in other words the concentration of the analyte.

    02:23 In immunoprecipitation, there is a mixture of antigens with the antigen of interest but also other antigens.

    02:33 A excess of immobilized antibody specific for the antigen of interest is added.

    02:41 This causes precipitation when the antigen of interest is bound by the antibody, immobilized onto for example a particle. And these immobilized antibodies bound to the antigen of interest can then be deposited at the bottom of a test tube using centrifugation. These can then be washed with a fresh solution to remove any unbound antigen. And then the antibody denatured to release or elute the antigen of interest, thereby isolating that specific antigen from a mixture of antigens. In radial immunodiffusion, there is an agarose gel containing specific polyclonal antibody. Wells are cut into the gel and the test sample added to one of the wells. There are also standards with defined concentrations of the antigen added to the gel. After 24-48 hours, precipitant rings will be produced. As the analyte diffuses into the gel, specific antibody binds and the precipitate forms. The size of the precipitation ring depends on the quantity of analyte loaded.

    About the Lecture

    The lecture Immunodiagnostic Methods: Agglutination, Nephelometry, Immunoprecipitation and Radial Immunodiffusion by Peter Delves, PhD is from the course Immunodiagnostics. It contains the following chapters:

    • Overview of Immunodiagnostic Methods
    • Agglutination
    • Nephelometry
    • Immunoprecipitation
    • Radial Immunodiffusion

    Included Quiz Questions

    1. Plasma proteins
    2. Red blood cells
    3. White blood cells
    4. Platelets
    5. Plasma volume
    1. Isolating a specific antigen from a mixture of antigens
    2. Detecting a specific antigen in a sample
    3. Quantifying protein levels in CSF
    4. Quantification of a specific antigen using a gel that contains antibodies
    5. Detecting a specific antibody in a sample

    Author of lecture Immunodiagnostic Methods: Agglutination, Nephelometry, Immunoprecipitation and Radial Immunodiffusion

     Peter Delves, PhD

    Peter Delves, PhD

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