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    ELISA Technique

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    najdat alzaatra
    ELISA (Enzyme-Linked Immunosorbent Assay) is a laboratory technique that uses antibodies and antigen-enzyme conjugates to detect and quantify substances like proteins in a biological sample. There are two main types of ELISA - competitive ELISA and sandwich ELISA. In competitive ELISA, a labelled antigen competes with the sample antigen for binding sites on an antibody, while in sandwich ELISA the ELISA plate is coated with an antibody to capture a specific antigen which is then detected with a secondary antibody-enzyme conjugate. The presence and quantity of the antigen in samples can then be determined by measuring the enzymatic activity.

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    ELISA Technique

    Uploaded by

    najdat alzaatra
    21 views19 pages
    ELISA (Enzyme-Linked Immunosorbent Assay) is a laboratory technique that uses antibodies and antigen-enzyme conjugates to detect and quantify substances like proteins in a biological sample. There are two main types of ELISA - competitive ELISA and sandwich ELISA. In competitive ELISA, a labelled antigen competes with the sample antigen for binding sites on an antibody, while in sandwich ELISA the ELISA plate is coated with an antibody to capture a specific antigen which is then detected with a secondary antibody-enzyme conjugate. The presence and quantity of the antigen in samples can then be determined by measuring the enzymatic activity.

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    Original Title

    12. ELISA Technique

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    ELISA (Enzyme-Linked Immunosorbent Assay) is a laboratory technique that uses antibodies and antigen-enzyme conjugates to detect and quantify substances like proteins in a biological sample. There are two main types of ELISA - competitive ELISA and sandwich ELISA. In competitive ELISA, a labelled antigen competes with the sample antigen for binding sites on an antibody, while in sandwich ELISA the ELISA plate is coated with an antibody to capture a specific antigen which is then detected with a secondary antibody-enzyme conjugate. The presence and quantity of the antigen in samples can then be determined by measuring the enzymatic activity.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    21 views19 pages

    ELISA Technique

    Uploaded by

    najdat alzaatra
    ELISA (Enzyme-Linked Immunosorbent Assay) is a laboratory technique that uses antibodies and antigen-enzyme conjugates to detect and quantify substances like proteins in a biological sample. There are two main types of ELISA - competitive ELISA and sandwich ELISA. In competitive ELISA, a labelled antigen competes with the sample antigen for binding sites on an antibody, while in sandwich ELISA the ELISA plate is coated with an antibody to capture a specific antigen which is then detected with a secondary antibody-enzyme conjugate. The presence and quantity of the antigen in samples can then be determined by measuring the enzymatic activity.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 19

    ELISA

    Enzyme Linked Immunosorbent Assay


    Definitions

     Antibodies (also known as immunoglobulins


    abbreviated Ig) are gamma globulin proteins that
    are found in blood and are used by the immune
    system to identify and neutralize foreign objects,
    such as bacteria and viruses.
    Definitions- cont
     Antigens
    A substance that when introduced into the body
    stimulates the production of an antibody

     Immunoassay
    A laboratory technique that makes use of the
    binding between an antigen and its
    homologous antibody in order to identify and
    quantify the specific antigen or antibody in a
    sample
    Definitions- cont
     Analyte

    The sample being analyzed and in


    immunoasssays the analyte is either Antibody
    or Antigen
    Introduction
     The Antibody: An immunoglobulin, a
    specialized immune protein, produced because
    of the introduction of an antigen into the body,
    and which possesses the remarkable ability to
    combine with the very antigen that triggered its
    production (specific affinity)

     The antibody recognises and bind to the


    antigenic determinant region of the antigen
    Competitive ELISA

     The labelled antigen competes for primary


    antibody binding sites with the sample antigen
    (unlabeled). The more antigen in the sample,
    the less labelled antigen is retained in the well
    and the weaker the signal).
     https://www.youtube.com/watch?v=Kb26nQV
    MHds
    Sandwich ELISA
     TheELISA plate is coated with Antibody to
    detect specific antigen
    Sandwich ELISA

     Preparea surface to which a known quantity of


    capture a primary antibody is bound.

     Blockany non specific binding sites on the


    surface

     Apply the antigen-containing sample to the


    plate.
    Sandwich ELISA-Cont

     Wash the plate, so that unbound antigen is


    removed.

     Apply enzyme linked secondary antibodies as


    detection antibodies which also bind specifically
    to the antigen.

     Washthe plate, so that the unbound antibody-


    enzyme conjugates are removed.
    Sandwich ELISA-Cont
     Applya chemical which is converted by the
    enzyme into a coloured product.

     Measure the absorbency of the plate wells to


    determine the presence and quantity of antigen
    Sandwich ELISA
    An example of an ELISA experiment
     Before starting the work read kit instruction
    carefully

     1-The 96 well plate is labeled carefully and


    the first wells are used to draw the standard
    curve
    An example of an ELISA experiment-Cont

     The sample is added to plate in duplicate or


    triplicate and then the mean result is calculated

     Thequality control sample which is provided


    with the kit is treated as the test samples
    Results
     After
    reading the results the standard curve is
    drawn were the concentration is blotted on the
    X-axis and the absorbance on the Y-axis

    Absorption
    nm

    Concentration ng/ml
    Results-cont
     The standards concentrations is specified on
    the x-axis and the reading of each standard is
    specified on the y-axis and the standard curve
    is drawn
    Results-cont
     This standard curve is used to determine the
    unknown concentration of each sample by finding
    the opposite concentration to the absorbance

     https://www.youtube.com/watch?v=0c2FdQH33hk
     https://www.youtube.com/watch?v=cHSVmzZ84FU
     https://www.youtube.com/watch?v=zR_xlV5v_f4

    Absorption
    nm

    Concentration ng/ml

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