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    ELISA

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    Ahmed Nagy
    ELISA is an enzyme-linked immunosorbent assay used to detect substances like proteins, hormones, antibodies, and drugs. It works by binding an antigen to a solid surface and detecting it using an antibody linked to an enzyme. There are direct, indirect, sandwich, and competitive ELISA types that differ in how the antigen and antibodies are arranged. ELISA is a sensitive, specific, and efficient quantitative analysis technique that requires small sample volumes and has many applications like detecting diseases, allergens, and antibody concentrations.

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    ELISA

    Uploaded by

    Ahmed Nagy
    45 views19 pages
    ELISA is an enzyme-linked immunosorbent assay used to detect substances like proteins, hormones, antibodies, and drugs. It works by binding an antigen to a solid surface and detecting it using an antibody linked to an enzyme. There are direct, indirect, sandwich, and competitive ELISA types that differ in how the antigen and antibodies are arranged. ELISA is a sensitive, specific, and efficient quantitative analysis technique that requires small sample volumes and has many applications like detecting diseases, allergens, and antibody concentrations.

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    presentation about the ELISA technique

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    ELISA is an enzyme-linked immunosorbent assay used to detect substances like proteins, hormones, antibodies, and drugs. It works by binding an antigen to a solid surface and detecting it using an antibody linked to an enzyme. There are direct, indirect, sandwich, and competitive ELISA types that differ in how the antigen and antibodies are arranged. ELISA is a sensitive, specific, and efficient quantitative analysis technique that requires small sample volumes and has many applications like detecting diseases, allergens, and antibody concentrations.

    Copyright:

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

    ELISA

    Uploaded by

    Ahmed Nagy
    ELISA is an enzyme-linked immunosorbent assay used to detect substances like proteins, hormones, antibodies, and drugs. It works by binding an antigen to a solid surface and detecting it using an antibody linked to an enzyme. There are direct, indirect, sandwich, and competitive ELISA types that differ in how the antigen and antibodies are arranged. ELISA is a sensitive, specific, and efficient quantitative analysis technique that requires small sample volumes and has many applications like detecting diseases, allergens, and antibody concentrations.

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    Molecular Biology

    Bio-321
    ELISA
    Enzyme-Linked Immuno-Sorbent Assay
    LAB 2
    ❖ What is ELISA?
    It stands for Enzyme-linked immunosorbent assay (measurement), a rapid
    immunochemical test that involves an enzyme used for measuring a wide variety of tests
    of body fluids.

    ✓ ELISA is considered to be a quantitative analysis .

    ✓ It is a solid phase (plate based assay).


    ❖ Uses
    ELISA tests detect substances that have antigenic properties:
    • primarily large molecules as; Proteins , growth factors, inflammatory factors and
    Hormones.
    • Small molecules and ions, such as glucose and potassium.
    • Some of other substances as; Bacterial Antigens, Antibodies and Drugs
    ❖ Principle
    The test depends on the interaction between components of the human body
    (Antigen) with an (Antibody)
    An antigen binds to a solid surface, then recognized specifically by an antibody
    conjugated with an enzyme.

    After then it is incubated with the enzyme substrate to give a measurable product since
    color intensity reflect amount of antigen measured

    N.B:

    1. Antigens are the substances to which antibodies are produced as they stimulate
    an immune response.

    2. Antibodies are proteins produced by the body to identify and neutralize any foreign
    substances that may be encountered, such as viruses and bacteria.
    ❖ Advantages
    1) Fast (many samples can be processed at once).

    2) ELISA tests are generally highly sensitive and specific

    3) Very little sample volume is needed( <10 μl in most cases)

    4) Easy to learn (simple procedures)


    ❖ Basic immunological protocols :

    Immobilization of antigen\ antibody to solid surface


    96 well or 384 well polystyrene plates (microplates)
    Addition of reagents
    Specific binding mostly via antigen antibody reaction
    Washing steps
    Separation bound from nonbounded material elimination of non
    specifically bound material as excess antibody or antigen added
    ELISA Types

    D) Special:
    A) Direct B) Indirect C) Sandwich
    Competitive
    A. DIRECT ELISA
    It is a direct detection involve attachment of the antigen (sample) to the solid
    phase followed by primary antibody is conjugated with the enzyme to give a
    measurable product for antigen detection
    B. INDIRECT ELISA
    It is an indirect detection also involve attachment of antigen to the solid phase but the
    primary antibody is not labelled as in the direct ELISA method then we add labeled a
    secondary enzyme conjugated antibody to the primary antibody bound to antigen to
    be detected.
    C . sandwich ELISA
    It is more efficient in signal
    amplification, rapid, easy , highly specific
    principle: we measure the antigen (sample)
    between two layers of antibodies (capture and
    detection antibody).
    Capture antibody is attached to a solid phase
    support then adding antigen samples with a
    buffer to minimize attachment to solid phase
    then an enzyme labelled antibody is added for
    detection
    The target antigen must contain at least two
    antigenic sites capable of binding to antibodies.
    D . Special ELISA
    competitive ELISA
    (inhibition ELISA)
    principle: labelling a purified antigen instead of the
    antibody since competition between unlabeled antigen
    (sample) and the labelled antigen (reference) for binding to
    the capture antibody common in small antigens of only one
    binding site.
    • competitive ELISA assays measure the concentration of an
    antigen by detection of signal interference.
    • The sample antigen competes with a reference antigen for
    binding to a specific amount of labeled antibody.
    • The reference antigen is pre-coated on a multi-well plate
    and sample is pre-incubated with labeled antibody and
    added to the wells.
    • SO Depending on the amount of antigen in the sample, more or less free
    antibodies will be available to bind the reference antigen. This means the more
    antigen there is in the sample, the less reference antigen will be detected and the
    weaker the signal.
    N.B
    Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The
    labeled antigen and the sample antigen (unlabeled) compete for binding to the
    primary antibody. The lower the amount of antigen in the sample, the stronger the
    signal due to more labeled antigen in the well.
    Application of ELISA

    ❑Presence of antigen or the presence of antibody in


    a sample can be evaluated.
    ❑Determination of serum antibody concentrations
    in a virus test.
    ❑used in food industry when detecting potential
    food allergens.
    ❑Applied in disease outbreaks- tracking the spread
    of disease e.g. HIV, bird flu, common, colds,
    cholera, STD etc.
    SUM UP:
    Thank You

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