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    Elisa Biology Project 12TH

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    srimannivi
    ELISA is an assay that detects the presence of antigens or antibodies in a blood sample. It works by binding an antigen or antibody to a plate, then using a secondary antibody linked to an enzyme to detect the first bound antibody. The enzyme produces a colored product that can be read by a spectrophotometer, indicating the presence of the target antigen or antibody. ELISA is a sensitive technique used in various applications like detecting infections, hormones, allergies, and screening blood donations.

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    Elisa Biology Project 12TH

    Uploaded by

    srimannivi
    36 views8 pages
    ELISA is an assay that detects the presence of antigens or antibodies in a blood sample. It works by binding an antigen or antibody to a plate, then using a secondary antibody linked to an enzyme to detect the first bound antibody. The enzyme produces a colored product that can be read by a spectrophotometer, indicating the presence of the target antigen or antibody. ELISA is a sensitive technique used in various applications like detecting infections, hormones, allergies, and screening blood donations.

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    Biology elisa

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    ELISA BIOLOGY PROJECT 12TH

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    ELISA is an assay that detects the presence of antigens or antibodies in a blood sample. It works by binding an antigen or antibody to a plate, then using a secondary antibody linked to an enzyme to detect the first bound antibody. The enzyme produces a colored product that can be read by a spectrophotometer, indicating the presence of the target antigen or antibody. ELISA is a sensitive technique used in various applications like detecting infections, hormones, allergies, and screening blood donations.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    36 views8 pages

    Elisa Biology Project 12TH

    Uploaded by

    srimannivi
    ELISA is an assay that detects the presence of antigens or antibodies in a blood sample. It works by binding an antigen or antibody to a plate, then using a secondary antibody linked to an enzyme to detect the first bound antibody. The enzyme produces a colored product that can be read by a spectrophotometer, indicating the presence of the target antigen or antibody. ELISA is a sensitive technique used in various applications like detecting infections, hormones, allergies, and screening blood donations.

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    © All Rights Reserved
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    of 8

    ELISA

    ENZYME LINKED IMMUNOSORBENT ASSAY


    AIM/OBJECTIVE: To detect the presence of foreign antigen or antibody
    produced against the antigen in the blood.

    DEFINITION: The Enzyme Linked ImmunoSorbent Essay (ELISA) is a common


    laboratory technique which is used to measure the concentration of an analyte
    (usually antibodies or antigen) in solution

    WHY KNOWN AS …. ? ENZYME LINKED IMMUNOSORBENT


    ASSAY
    1. Antigen /antibody of interest is absorbed on to the plastic surface (“sorbent”).

    2. Antigen recognized by specific antibody (“immune”).

    3. The antibody is recognized by second antibody (“immune”) which the enzyme


    has attached (“enzyme linked”).

    4. Substrate reacts with the enzyme to produce product, usually colored.


    HISTORY OF ELISA:
    Radioimmunoassay was first described paper rasalyn sussman yalow and Solomon
    berson published in 1960.

    In 1971 Peter perlmann and Eva engvall at Stockhlom University in Sweden and
    Anton schuurs and beuke van weemen in the Netherlands independently published
    papers that synthesized this knowledge into methods to perform EIA/ELISA.

    BASIC TERMS
    ➢ SOLID PHASE:
    Usually a micro titer plate well having 8 rows and 12 columns well format.

    ➢ ABSORPTION:
    The process of adding an antigen /antibody, diluted in buffer, so it attaches
    to the solid phase on incubation.
    ➢ WASHING: The simple flooding & emptying of wells with a buffered
    solution to separate bound from un-bound reagent in ELISA.
    ➢ ANTIGEN: Any molecule that elicits the production on antibodies when
    introduced into body.
    ➢ ANTIBODIES: Proteins produced in response to antigenic stimuli.
    ➢ ENZYME CONJUGATE: An enzyme that is attached irreversibly to an
    antibody.
    e.g.: horse reddish peroxide (HRPO).
    ➢ CHROMOGEN : A chemical alters color as a result of an enzyme
    interaction with substrate (color reaction used as signal) e.g.: trimethyl
    benzidine (TMB)
    ➢ STOPPING: The process of stopping the action of an enzyme on a
    substrate.
    ➢ READING: Spectrophotometric measurement of color developed in
    ELISA.

    Principle of Elisa
    • Based on immunology response
    • Lock and key concept
    1. Antigen (key) 2. Antibody (lock) key fits into lock
    • Enzyme conjugate substrate.
    Equipments
    1. MICROWELL PLATE: Flat bottom polystyrene plate contains 8rows and
    12 column wells holding 350 micro litres each.

    2. MULTI PIPETTE: An 8 channel 100 micro litres pipette is a good help for
    even small scale work.

    3. WASHING DEVICE: May be of use particularly when there is a risk that the
    samples tested in ELISA contain infectious material so must be collected for
    subsequent disinfection.

    4. MICROPLATE WASHER: There are very efficient with unusually low


    carry over contamination.
    microplate washer washing device microplate pipette

    DIFFERENT TYPES OF ELISA:


    Reading: Measure the absorbent at 450nm with the help of ELISA reader.
    Calculate the absorbance for each sample and reference. Ascent software for the
    calculation of result can be used.
    OBERVATION:
    SAMPLE COLOUR

    Positive control Dark purple


    Negative control Light green
    Patient A Light green
    Patient B green
    Patient C Dark purple

    ANALYSIS:

    RESULT:
    SAMPLES OF PATIENTS TEST RESULTS

    Patient A NEGATIVE

    Patient B INTERMEDATE

    patient C POSITIVE

    PRECAUTIONS
    • Use of exchange type pipette. (Always use new tip).Wash the solid phase
    (ELISA plate) properly before and after the experiment.
    • Use reservoir for each reagent and label the reservoir. Don’t use the same
    reservoir for multiple reagents. Don’t return the reagent to the stock.
    • During incubation well plated should be covered using plate cover. Plate
    cover s efficient only under suitable conditions (room temperature)
    humidity > 50 % air steam < 0.2 m/sec.
    • Coated of wells should be proper with the addition of blocking solution.
    • Improper coating leads to false positive results.

    ADVANTAGES OF ELISA
    • Reagents are relatively cheap & have long life.
    • It is highly specific &sensitive (<1pg/ml)
    • Easy to perform and quick procedure.
    • Equipments is widely available
    • It can be used on most type of biological sample like plasma, serum, urine,
    cell extraction.
    • No radiation hazards occur during labeling or disposal of waste.

    Disadvantages of Elisa
    • Measurement of enzyme activity can be more complex than the
    measurement of activity of some type of radioisotopes
    • False positive/negative possible, especially with mutated/altered
    antigen
    • Enzyme activity may be affected by plasma constituents
    • Very specific to particular antigen so it won’t recognize other
    antigen.

    Application
    ➢ Screening donated blood for evidence of viral contamination by
    ✓ HIV-1 and HIV-2(presence of anti-HIVantibodies)
    ✓ Hepatitis C (presence of antibodies)
    ✓ Hepatitis B (testing for both antibodies and a viral antigen)
    ➢ Measuring hormone level
    ✓ HCG( as a test for pregnancy)
    ✓ LH (determining the time of ovulation)
    ✓ TSH,T3 and T4 ( for thyroid function)
    ➢ Detecting infection
    ✓ Sexually transmitted agents like HIV, syphilis and chlamydiasis.
    ✓ Hepatitis B and C
    ✓ Toxoplasma gondii
    ➢ Detecting illicit drugs
    ➢ Detecting allergies in food and house dust.

    Bibliography
    • www.google.co.in
    • www.wikipedia.com

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