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    ELISA

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    AAnneMichelleP.Cruz
    ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of an antibody or antigen in a sample. It involves using antibodies and color change to identify a substance. The first ELISA was developed in the 1970s as a replacement for radioimmunoassays. In an ELISA, an antibody or antigen is attached to a polystyrene plate and an enzyme-linked antibody is added to detect if the target is present, resulting in a color change. There are different types of ELISA including direct, indirect, sandwich, and competitive, which vary in their detection methods. ELISA has many applications such as detecting hormones, bacteria, viruses, and toxins.

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    ELISA

    Uploaded by

    AAnneMichelleP.Cruz
    185 views32 pages
    ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of an antibody or antigen in a sample. It involves using antibodies and color change to identify a substance. The first ELISA was developed in the 1970s as a replacement for radioimmunoassays. In an ELISA, an antibody or antigen is attached to a polystyrene plate and an enzyme-linked antibody is added to detect if the target is present, resulting in a color change. There are different types of ELISA including direct, indirect, sandwich, and competitive, which vary in their detection methods. ELISA has many applications such as detecting hormones, bacteria, viruses, and toxins.

    Original Description:

    talks about the Enzyme-linked Immunosorbent Assay and its types

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    © © All Rights Reserved

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    ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of an antibody or antigen in a sample. It involves using antibodies and color change to identify a substance. The first ELISA was developed in the 1970s as a replacement for radioimmunoassays. In an ELISA, an antibody or antigen is attached to a polystyrene plate and an enzyme-linked antibody is added to detect if the target is present, resulting in a color change. There are different types of ELISA including direct, indirect, sandwich, and competitive, which vary in their detection methods. ELISA has many applications such as detecting hormones, bacteria, viruses, and toxins.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    185 views32 pages

    ELISA

    Uploaded by

    AAnneMichelleP.Cruz
    ELISA (enzyme-linked immunosorbent assay) is a biochemical technique used to detect the presence of an antibody or antigen in a sample. It involves using antibodies and color change to identify a substance. The first ELISA was developed in the 1970s as a replacement for radioimmunoassays. In an ELISA, an antibody or antigen is attached to a polystyrene plate and an enzyme-linked antibody is added to detect if the target is present, resulting in a color change. There are different types of ELISA including direct, indirect, sandwich, and competitive, which vary in their detection methods. ELISA has many applications such as detecting hormones, bacteria, viruses, and toxins.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
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    ELISA was developed in the 1970s as a replacement for radioimmunoassays. It uses antibodies and enzymes to detect antigens or antibodies in a sample by producing a measurable color change.

    ELISA was first developed in the early 1970s as a replacement for radioimmunoassays by Avramais and Pierce who developed methods to chemically link antibodies to biological enzymes whose activities produce a measurable signal with solutions containing appropriate substrates.

    ELISA is a test that uses antibodies and a change in color to identify a substance. It detects if a certain antigen or antibody is present in a patient using an enzyme. It is an immunologic test to determine levels of protein in a biological sample.

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    HISTORY
    First developed in the early 1970s as a
    replacement for radioimmunoassays.
    Avramais and Pierce developed
    methods to chemically link antibodies
    to biological enzymes whose activities
    produce a measurable signal with
    solutions containing appropriate
    substrates.

    PRINCIPLE
    A test that used to identify a substance using
    antibodies and change in color.
    Used to detect if there is a certain antigen or
    antibody is present in a patient using an
    enzyme
    An immunologic test to determine levels of
    protein in a biological sample.

    ELISA
    EL

    Enzyme-linked

    : Enzymes activity is used as a


    reporter
    : The enzymatic reaction will
    produce a colored species

    ELISA
    EL

    Enzyme-linked

    IS

    Immunosorbent

    : An antibody or antigen (immuno) is


    adsorbed (sorbent) onto the
    polystyrene wells in which we conduct the
    test.

    ELISA
    EL

    Enzyme-linked

    IS

    Immunosorbent

    Assay

    : We will could determine the amount of NPTII


    (quantitative assay).
    : We will use NPTII concentration standards to
    construct a standard curve

    ENZYMES USED IN ELISA

    Horseradish Peroxidase

    ENZYMES USED IN ELISA

    Alkaline Phosphatase

    MICROTITER PLATE

    Is a flat plate made of polystyrene with multiple wells used


    as small test tubes. It has become a standard tool in
    analytical research and clinical diagnostic testing
    laboratories.

    ELISA READER / MICROPLATE


    READERS

    - Is integrally installed in a personal computer


    - It is where the plate is stably positioned in the
    carrier and between the light source board and the
    scanning assembly, and scanned by the scanning

    Materials needed
    for ELISA Kit:
    ELISA Plate
    Positive Control
    Negative Control
    Dilution buffer
    Conjugate
    TMB substrate
    Stop solution

    A
    S
    I
    L
    E
    F
    O
    S
    E
    P
    TY

    DIRECT
    ELISA
    Involve attachment
    of the antigen to
    the polystyrene
    plate followed by
    an enzyme labeledantibody

    ADVANTAGES
    Quick methodology
    Cross-reactivity of secondary
    anitbody is eliminated

    DISADVANTAGES
    Must be labeled individually
    Time- consuming
    Inflexible when performing multiple experiments
    The signal is less amplified (lower sensitivity)
    Immunoreactivity of the primary anitbody may be
    reduced as a result of labeling

    INDIRECT
    ELISA
    Involves attachment of
    the antigen to the
    polystyrene plate, but in
    this case, the primary
    antibody is not labeled.
    An enzyme conjugated
    secondary antibody,
    directed at the first
    antibody, is then added.

    ADVANTAGES
    Immunoreactivity of primary antibody is
    not affected by labeling
    Wide variety of secondary antibodies
    available on the market
    More signal amplification (Sensitivity is
    increased)

    DISADVANTAGES
    Cross-reactivity may occur with the secondary
    antibody, resulting in nonspecific signal.
    An extra incubation step is required in the
    procedure.

    SANDWICH ELISA
    Measures the amount of
    antigen between two
    layers of antibodies. The
    antigens to be
    measured must contain
    at least two antigenic
    sites, capable of binding
    to the antibody, since at
    least two antibodies act
    in the sandwich.

    4 factors where
    sensitivity depends:
    1. Number of molecules of the first antibody
    that are bound to the solid phase
    2. Avidity of the second antibody to the
    antigen
    3. Avidity of the second antibody to the
    antigen
    4. Specific activity of the second antibody

    ADVANTAGES
    High specificity, since two antibodies are used
    the antigen/analyte is specifically captured and
    detected
    Suitable for complex samples, since the antigen
    does not require purification prior to
    measurement
    Flexibility and sensitivity, since both direct and
    indirect detection methods can be used

    DOT ELISA

    COMPETITIVE
    ELISA
    This is the most complex
    ELISA, and is used to
    measure the
    concentration of an
    antigen (or antibody) in a
    sample by observing
    interference in an
    expected signal output.
    Hence, it is also referred
    to as an inhibition ELISA.

    REVERSE ELISA
    A new tecnique that uses a strong stage created up of an
    immunosorbent polystyrene rod with 4-12 sticking out
    ogives.
    The whole system is engrossed in a test tube containing
    the gathered example and the following actions (incubation
    in conjugate, washing and incubation in chromogenous) are
    performed by dropping the ogives in microwells of
    conventional micro plates pre-filled with reagents.

    ADVANTAGES
    The ogives can every be sharpened to an alternate reagent,
    permitting the concurrent identification of distinctive antibodies
    and diverse antigens for multi-target assays
    The specimen volume could be expanded to enhance the test
    affectability in clinical, sustenance and ecological tests; One
    ogive is left unsensitized to measure the non-particular
    responses of the example
    The utilization of research center supplies for apportioning
    specimen aliquots, washing result and reagents in microwells is
    not needed, expediting prepared to-utilize lab-kits.

    MULTIPLEX
    ELISA
    Logical progression of
    the widely used
    microtiter plate ELISA is
    toward a protein array
    format that allows
    simultaneous detection
    of multiple analytes a
    multiple array addresses
    withtin a single well.

    ADVANTAGES (in general)


    More accurate
    High sensitivity
    High specificity
    Does not need radioisotopes
    can be used for a variety of infections
    No radiation hazards occur during labelling and
    disposal of waste

    DISADVANTAGES
    It requires skilled laboratory professionals and
    specific research facility types of equipments
    More complex measurements of enzyme
    activity
    Plasma constituents effect on enzyme activity

    Applications of ELISA:
    Useful device both for identifying serum
    antibody levels (such as with the HIV analyze or
    West Nile Virus) and also for discovering the use
    of antigen.
    It has also found applications in the food
    industry in detecting potential food substances
    such as dairy products, walnuts, peanuts,
    almonds, and egg.

    Measuring hormone levels (HCG; LH; TSH, T3,


    and T4)
    Detects many bacterial and viral antigens
    Used in toxicology as a rapid presumptive
    screen for certain classes of drugs.
    Detect various kind of diseases, such as
    malaria, Chagas disease, and Johne's disease.

    Other applications:
    detection of Mycobacterium antibodies in
    tuberculosis
    detection of rotavirus in feces
    detection of hepatitis B markers in serum
    detection of enterotoxin of E. coli in feces

    Sources:
    http://history.cpet.ufl.edu/lm/elisa/types01.html
    https://labtestsonline.org/understanding/analytes/alp/tab/te
    st
    /
    http://www.elisa-antibody.com/ELISA-Introduction/elisaadvantages
    https://lifescienceproduct.wordpress.com/2013/09/14/elisa/
    http://www.slideshare.net/AnitaSingh13/elisa-ppt

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