Elaeza project for education only not.pptx

Elaeza project for education only not.pptx

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  ELISA PRESENTED BY -TAPASWINI PARIDA ROLL NO CLASS GUIDED B Y - BS(B)22-073 - +3 3r YEAR - SASMITA BARAL & SABY ASACHI T ANA Y A NEW STAR DEGREE SCIENCE COLLEGE PUBUSAHI, KHORDHA
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  ELISA • Enzyme-linked immunosorbentassay is commonly known as ELISA where Ag- Ab interaction is monitored by enzyme measurement. • It is similar in principle to Radio Immuno Assay (RIA) but depends 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.
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  PRINCIPLE • ELISA usean enzyme to detect the binding of antigen (Ag) antibody (Ab). • The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding. • An ELISA can be used to detect either the presence of antigens or antibodies in a sample depending how the test is designed.
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  • A numberof enzymes have been employed for ELISA, including 1. Alkaline phosphatase 2. Horseradish peroxidase 3. Galactosidase • These assays approach the sensitivity of RIAs and have the advantage of being safer and less costly.
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  Variants Of ELISA •A number of variations of ELISA have been developed, allowing qualitative detection or quantitative measurement of either antigen or antibody. • Each type of ELISA can be used qualitatively to detect the presence of antibody or antigen. • Alternatively, a standard curve based on known concentrations of antibody or antigen is prepared, from which the unknown concentration of a sample can be determined.
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  INDIRECT ELISA • Antibodycan 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 antibody (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.
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  • Any freeAb₂ then is washed away and a substrate for the enzyme is added. • The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds.
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  • Indirect ELISAdetects the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS. • 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 infection.
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  SANDWICH ELISA • Antigencan 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.
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  • After anyfree second antibody is removed by washing, substrate is added, and the colored reaction product is measured.
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  COMPETITIVE ELISA • Anothervariation 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.
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  • Addition ofan 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 an indirect ELISA. • In the competitive assay however, higher the concentration of antigen in the original sample, the lower will be the absorbance.
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  APPLICATION • Serum antibodyconcentration. • Detecting potential food allergens. (milk, peanut, walnut, almonds and eggs) • Tracking the spread of disease in disease outbreaks. (e.g. HIV , bird flu, common COLD, STDs.) • Detection of antigen. (e.g. pregnancy hormones, drug allergens, GMO, mad cow disease) • Detection of antibodies to past exposure to disease. (e.g. Lyme disease, trichinosis, HIV , bird flu)