Scribd Logo
Upload

Welcome to Scribd!

  • Exp 5

    Uploaded by

    Hema Kannan
    102 views3 pages
    This document describes an experiment using the ELISA technique to study the interaction between antigens and antibodies. The objective was to study ELISA and antigen-antibody interactions. Wells of a microtiter plate were coated with antigens and exposed to antibodies, then a secondary antibody linked to an enzyme. Absorbance readings of the wells after adding a substrate varied, likely due to insufficient washing, temperature variations during incubation, or contamination from reusing pipette tips. ELISA allows quantifying the amount of antigen or antibody through measuring the color intensity, proportional to enzyme concentration, produced in the antigen-antibody complex.

    Original Description:

    Copyright

    © Attribution Non-Commercial (BY-NC)

    Available Formats

    DOCX, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document
    This document describes an experiment using the ELISA technique to study the interaction between antigens and antibodies. The objective was to study ELISA and antigen-antibody interactions. Wells of a microtiter plate were coated with antigens and exposed to antibodies, then a secondary antibody linked to an enzyme. Absorbance readings of the wells after adding a substrate varied, likely due to insufficient washing, temperature variations during incubation, or contamination from reusing pipette tips. ELISA allows quantifying the amount of antigen or antibody through measuring the color intensity, proportional to enzyme concentration, produced in the antigen-antibody complex.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    102 views3 pages

    Exp 5

    Uploaded by

    Hema Kannan
    This document describes an experiment using the ELISA technique to study the interaction between antigens and antibodies. The objective was to study ELISA and antigen-antibody interactions. Wells of a microtiter plate were coated with antigens and exposed to antibodies, then a secondary antibody linked to an enzyme. Absorbance readings of the wells after adding a substrate varied, likely due to insufficient washing, temperature variations during incubation, or contamination from reusing pipette tips. ELISA allows quantifying the amount of antigen or antibody through measuring the color intensity, proportional to enzyme concentration, produced in the antigen-antibody complex.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    of 3

    UNIVERSITY TUNKU ABDUL RAHMAN FACULTY OF SCIENCE

    Subject Code Subject Title Year/Semester : UDEE3214 : IMMUNOLOGY : YEAR3 SEMESTER1

    Experiment Title: ELISA

    Instructor: Ms.Norliza

    Marks:

    DECLARATION I Mr / Ms SUGANTHI PAKIANATHAN here by certify that this

    assignment is my original work and materials used from other sources have been properly acknowledged.

    Name: Suganthi Pakianathan Student ID No.: 0907508 Assessors Feedback:

    Signature: ____________ Date: 25 July 2011

    Objective(s): 1. To study on Enzyme-Linked Immunosorbent Assay (ELISA) 2. To study the interaction between antigen and antibody

    Results:

    Well 1 2 3 4 5 6 7 8

    Absorbance (A) 0.4868 0.3337 0.4261 0.4292 0.2796 0.4484 0.3728 0.4941

    Discussion: An antibody (Ab) reacts with the concerned antigen (Ag) in a highly specific manner for example an antibody reacts only with that determinant or region of an antigen for which it is specific; this produces an Ag-Ab complex. When soluble proteins react with an antibody, the Ag-Ab complex forms a precipitate, while in case of particulate antigens the Ag-Ab complex agglutinates.

    The antigen of interest is immobilized on the surface of a microtiter well. Specially constructed ELISA plates are routinely used for convenient handling of large numbers of test samples these plates have small wells at uniform intervals. Antibody specific to the antigen is added and allowed to react with the absorbed antigen. Unreacted molecules of the antibody are washed away leaving only the Ag-Ab complex. The antigen specific antibody is normal or unlabelled. This constitutes the primary reaction. In the secondary reaction an anti immunoglobulin is added into the vessel and allowed to react with the Ag-Ab complex already formed; the anti-Ig binds to the antibody component of the Ag-Ab complex. The anti-Ig is linked or conjugated to an appropriate enzyme molecule in such a way that its anti-Ig activity is not impaired. The unreacted anti-Ig is washed away and, finally, substrate of the enzyme is added along with the necessary reagents to develop colour due to the enzyme activity. The intensity of colour is proportional to the enzyme concentration; therefore, colour intensity is used to determine the quantity of antigen or antibody or simply to detect their presence. However, in our experiment the absorbance is fluctuating. This is because of insufficient of washing of wells may lead to the variations in absorbance. Other than that, variations in incubation temperature also may be the reason the result is fluctuating. Reuse of micropipette tip may be resulting in the presence of some residual which lead to contamination.

    References http://www.molecular-plant-biotechnology.info/recombinant-DNA-technology/enzymelinked-immunosorbent-assay-ELISA.htm

    You might also like