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    ELISA Lecture

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    querokeropi
    Labeled immunoassays use an enzyme, radioisotope, or fluorescent compound attached to an antibody or antigen to detect the presence of a protein like an antibody or antigen. Radioimmunoassays use radioactive labels like iodine-125 but are being replaced by enzyme immunoassays or fluorescent immunoassays due to health and safety issues. Enzyme immunoassays use an enzyme linked to an antibody or antigen to generate a detectable color change when it reacts with a substrate. They can be competitive or non-competitive and are used in tests like ELISA and RIA. Fluorescent immunoassays use fluorescent dyes to detect the presence of a protein through fluorescence microscopy or polarization.

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    ELISA Lecture

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    querokeropi
    146 views31 pages
    Labeled immunoassays use an enzyme, radioisotope, or fluorescent compound attached to an antibody or antigen to detect the presence of a protein like an antibody or antigen. Radioimmunoassays use radioactive labels like iodine-125 but are being replaced by enzyme immunoassays or fluorescent immunoassays due to health and safety issues. Enzyme immunoassays use an enzyme linked to an antibody or antigen to generate a detectable color change when it reacts with a substrate. They can be competitive or non-competitive and are used in tests like ELISA and RIA. Fluorescent immunoassays use fluorescent dyes to detect the presence of a protein through fluorescence microscopy or polarization.

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    Labeled immunoassays use an enzyme, radioisotope, or fluorescent compound attached to an antibody or antigen to detect the presence of a protein like an antibody or antigen. Radioimmunoassays use radioactive labels like iodine-125 but are being replaced by enzyme immunoassays or fluorescent immunoassays due to health and safety issues. Enzyme immunoassays use an enzyme linked to an antibody or antigen to generate a detectable color change when it reacts with a substrate. They can be competitive or non-competitive and are used in tests like ELISA and RIA. Fluorescent immunoassays use fluorescent dyes to detect the presence of a protein through fluorescence microscopy or polarization.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    146 views31 pages

    ELISA Lecture

    Uploaded by

    querokeropi
    Labeled immunoassays use an enzyme, radioisotope, or fluorescent compound attached to an antibody or antigen to detect the presence of a protein like an antibody or antigen. Radioimmunoassays use radioactive labels like iodine-125 but are being replaced by enzyme immunoassays or fluorescent immunoassays due to health and safety issues. Enzyme immunoassays use an enzyme linked to an antibody or antigen to generate a detectable color change when it reacts with a substrate. They can be competitive or non-competitive and are used in tests like ELISA and RIA. Fluorescent immunoassays use fluorescent dyes to detect the presence of a protein through fluorescence microscopy or polarization.

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    Attribution Non-Commercial (BY-NC)
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    Download as pptx, pdf, or txt
    of 31

    LABELED IMMUNOASSAY

    Characteristics of Labeled Assay


    1.

    2.
    3. 4. 5. 6.

    Competitive Noncompetitive Standards or calibrators Separation method solid phase Detection of label Quality control

    RADIOIMMUNOASSAY - RIA
    - Makes used of a radioactive substance 131I, 125I, 3H - It is easily incorporated into a protein molecule and it emits gamma radiation detected using a gamma counter - Thyroid function tests: TSH, FT3, FT4

    RIA
    Principle: Based on the principle of competitive binding where the unlabeled analyte being detected competes with a radiolabeled analyte for a limited number of binding sites on a solid-phase antibody

    disadvantage: - health hazard of radioactive substance - Disposal problem - Expensive equipment

    ELISA or EIA
    Designed to detect antigen or antibody by

    producing an enzyme-triggered color change It uses the catalytic properties of enzymes to detect and quantitate immunologic reactions Enzyme with its substrate detects the presence and quantity of antigen or antibody present in the patient specimen

    Enzymes
    Naturally occuring molecules that catalyze

    biochemical reactions They react with substrate that allows breakdown of products which could be chromogenic, fluorogenic, luminescent Can be measured qualitatively: detects presence of antigen or antibody quantitatively: by measuring the actual concentration of an analyte spectrophotometrically

    Various enzyme labels


    Horseradish peroxidase

    Alkaline phosphatase
    Glucose-6-phosphate dehydrogenase B-galactosidase

    Classification of EIA
    Heterogeneous - requires step to physically separate free from bound analyte 2. Homogeneous - no separation step necessary because enzyme activity diminishes when binding of antigen and antibody occurs
    1.

    Heterogeneous EIA
    Competitive b. Non-competitive c. Capture assay
    a.

    Non-competitive
    Indirect ELISA

    Has higher sensitivity


    Antigen or antibody attached to a solid phase HIV screening, HAV, HCV

    Principle of non-competitive EIA


    1. 2. 3. 4. 5.

    6.
    7. 8.

    Ag absorbed to the solid phase Serum that might contain the Ab is added Ag-Ab binding Washing Ab-enzyme conjugate added Enzyme-conjugated Ab will bind to the complex Washing Substrate is added

    Note: Amount of enzyme label detected is proportional to the amount of antibody in the specimen

    Indirect ELISA
    1.

    2.

    3. 4. 5. 6.

    7. 8.

    Ag absorbed to the solid phase Serum that might contain the Ab is added Ag-Ab binding Washing Ab-enzyme conjugate added Enzyme-conjugated Ab will bind to the complex Washing Substrate is added

    Competitive EIA
    Based on RIA

    Enzyme-labeled antigen competes with unlabeled

    patient antigen for a limited binding sites on antibody attached on a solid phase Used for measuring small antigens that are relatively pure: insulin and estrogen

    Principle of competitive EIA


    1. Labeled antigen competes with patient unlabeled antigen for a limited number of binding sites on solid-phase antibody. 2. Washing removes unbound antigen. Enzyme activity is determined. 3. Enzyme activity is inversely proportional to the concentration of the test substance: the more patient antigen is bound, the less enzyme-labeled antigen can attach.

    Capture Assay
    Sandwich immunoassay

    Antigen captured in this assay must have multiple

    epitopes Antibody is bound to a solid phase Used in measurement of immunoglobulins: IgM, IgE

    Principle

    Plate is coated with capture antibody 2. Sample is added and any antigen present binds to capture antibody 3. Enzyme-labeled secondary antibody is added and completes the sandwich 4. Substrate is added and is converted by enzyme to detectable form
    1.

    Note: Enzymatic activity is directly proportional to the amount of antigen in the test sample

    CAPTURE ASSAY
    Principle 1. Plate is coated with capture antibody 2. Sample is added and any antigen present binds to capture antibody 3. Enzyme-labeled secondary antibody is added and completes the sandwich 4. Substrate is added and is converted by enzyme to detectable form

    FLUORESCENT IMMUNOASSAY
    Makes use of fluorescent compounds:

    fluorophores or fluorochrome These compounds absorb energy from an incident light source and convert that energy into light of a longer wavelength and lower energy Measured in nanoseconds 2 compounds used: fluorescein and rhodamine

    1.

    Fluoroscein - absorbs maximally at 490 495nm - emits green color at 517 520nm - has high intensity, good photostability

    2. Tetramethylrhodamine absorbs at 550nm - emits red light at 580-585nm 3. Phycoerythrin 4. Europium 5. Lucifer yellow

    Use of Fluorescent Immunoassay: Histochemical localization of antigens in tissues: Immunofluorescent Assay - qualitative observation: fluorescence microscope - antigens can be detected in fixed tissue sections or in live cell suspension - antigen is determines by the appearance of a localized color against a dark background - for rapid identification of microorganism in cell culture or infected tissue, tumor-specific antigen on neoplastic tissue

    Fluorescent Staining:

    Direct immunofluorescent 2. Indirect immunofluorescent


    1.

    1.

    Direct immunofluorescent - antibody that is conjugated with a fluorescent tag is added directly to unknown antigen fixed on a microscope slide incubation. Washing - slide is read using fluorescence microscope - antigen is visualized as bright green apple or orange-yellow objects against dark background - best suited to antigen detection in tissue or body fluids

    2. Indirect immunofluorescent - involves 2 steps 1. incubation of patient serum with a known antigen attached to a solid phase washing. 2. antihuman immunoglobulin containing fluorescent tag is added

    Other Fluorescent Immunoassay

    3. FIA heterogenous and homogenous - similar to EIA 4. FPIA Fluorescence Polarization Immunoassay - based on the change in polarization fluorescent light emitted from a labeled molecule when it is bound by antibody - similar to competitive EIA

    CHEMILUMINESCENT IMMUNOASSAY
    Chemiluminescence

    - is the emission of light caused by a chemical reaction producing an excited molecule that decays back to its original ground state Substances used: luminol, acridinium esters, ruthenium derivatives, nitrophenyl oxalates
    - excellent sensitivity compared to EIA, RIA - faster turnaround time - used for immunologic testing

    Principle:

    Luminol or other substances used are oxidized using hydrogen peroxide and an enzyme.
    Intermediates are produced that are of higher energy state They return to their original state giving off energy in the form of light

    THANK YOU =)

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