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    Elisa 210503082334

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    Amanda
    The document discusses the ELISA technique, which uses enzymes to detect antibodies or antigens. It describes the basic principles of indirect, sandwich, and competitive ELISA. Common substrates and applications of ELISA are also mentioned.

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    Elisa 210503082334

    Uploaded by

    Amanda
    9 views12 pages
    The document discusses the ELISA technique, which uses enzymes to detect antibodies or antigens. It describes the basic principles of indirect, sandwich, and competitive ELISA. Common substrates and applications of ELISA are also mentioned.

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    elisa-210503082334

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    The document discusses the ELISA technique, which uses enzymes to detect antibodies or antigens. It describes the basic principles of indirect, sandwich, and competitive ELISA. Common substrates and applications of ELISA are also mentioned.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    9 views12 pages

    Elisa 210503082334

    Uploaded by

    Amanda
    The document discusses the ELISA technique, which uses enzymes to detect antibodies or antigens. It describes the basic principles of indirect, sandwich, and competitive ELISA. Common substrates and applications of ELISA are also mentioned.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
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    ELISA

    Dr. J. P. Saranraj
    ENZYME LINKED IMMUNOSORBANT
    ASSAY (ELISA)
     In 1972, Engvall and Perlmann discovered
    ELISA.
     Also called Enzyme Immunoassay (EIA) or
    Solid – phase Immunoassay.
     ELISA is similar in principle to RIA but differs on
    an Enzyme rather than a Radioactive label.
     An enzyme conjugated with an antibody
    reacts with a colorless substrate to generate a
    colored reaction product. Such a substrate is
    called a Chromogenic substrate.
     Number of enzymes have been employed for
    ELISA, including Alkaline phosphatase,
    1. Indirect ELISA
     Antibody can be detected or quantitatively
    determined with an Indirect ELISA.
     Serum or some other sample containing primary
    antibody (Ab1) is added to an antigen - coated
    microtiter well and allowed to react with the
    antigen attached to the well.
     After any free Ab1 is washed away, the presence
    of antibody bound to the antigen is detected by
    adding an enzyme-conjugated secondary anti-
    isotype antibody (Ab2), which binds to the
    primary antibody.
     Any free Ab2 then is washed away, and a
    Substrate for the Enzyme is added. The enzyme
    converts the substrate to a detectable colored
     The amount of colored reaction product that
    forms is measured by specialized
    spectrophotometric plate readers (ELISA
    Reader), which can measure the absorbance of
    all of the wells of a 96 -well plate in seconds.
     Indirect ELISA is the method of choice to detect
    the presence of serum antibodies against HIV.
     In this assay, recombinant envelope and core
    proteins of HIV are adsorbed as solid-phase
    antigens to microtiter wells. Individuals infected
    with HIV will produce serum antibodies to
    epitopes on these viral proteins.
     Generally, serum antibodies to HIV can be
    detected by indirect ELISA within 6 weeks of
    2. Sandwich ELISA
     Antigen can be detected or measured by a
    Sandwich ELISA.
     In this technique, the antibody (rather than the
    antigen) is immobilized on a microtiter well.
     A sample containing antigen is added and
    allowed to react with the immobilized antibody.
     After the well is washed, a second enzyme-
    linked antibody specific for a different epitope on
    the antigen is added and allowed to react with
    the bound antigen.
     After any free second antibody is removed by
    washing, substrate is added, and the colored
    reaction product is measured.
    3. Competitive ELISA
     Another variation for measuring amounts of
    antigen is competitive ELISA.
     In this technique, antibody is first incubated in
    solution with a sample containing antigen.
     The antigen-antibody mixture is then added to an
    antigen coated microtiter well.
     The more antigen present in the sample, the less
    free antibody will be available to bind to the
    antigen-coated well.
     Addition of an enzyme-conjugated secondary
    antibody (Ab2) specific for the isotype of the
    primary antibody can be used to determine the
    amount of primary antibody bound to the well as
     In the competitive assay, however, the higher the
    concentration of antigen in the original sample,
    the lower the absorbance.
    Substrates used in ELISA
     PNPP (p-Nitrophenyl Phosphate - Disodium Salt)
    is a widely used substrate for detecting Alkaline
    phosphatase.
     ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-
    sulfonic acid] - Diammonium salt) is used to
    detect Horse Radish Peroxidase.
     OPD (o-phenylenediamine dihydrochloride) is
    used to detect Horse Radish Peroxidase.
     TMB (3,3',5,5'-tetramethylbenzidine) used to
    detect Horse Radish Peroxidase.
    Applications of ELISA
     Detection of HIV antibodies in blood samples.
     Detection of Hepatitis - B markers in serum.
     Detection of Rotavirus in feces.
     Detection of Mycobacterium sp. antibodies in
    Tuberculosis.
     Detection of Enterotoxin of Escherichia coli in
    feces.
     Detect various kind of diseases such
    as Malaria, Chagas disease
    (Trypanosomiasis) and Johne's disease
    (Paratuberculosis – Mycobacterium avium).
     found applications in the food industry in
    detecting food allergens such

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