Enzyme-Linked Immunosorbent Assay or ELISA as it is more commonly known is an enzyme immunoassay (EIA), which is one of the most widely used applications of monoclonal antibodies.

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Enzyme-Linked Immunosorbent Assay or ELISA as it is more commonly known is an enzyme immunoassay (EIA), which is one of the most widely used applications of monoclonal antibodies.  Immunosorbent assays, as the term suggests, involve adsorption of either the antigen or the antibody to a surface, in most cases the bottom of a plastic well. The ELISA is used to detect and amplify an antigen-antibody reaction. It is the most basic test to determine if an individual is positive for a selected pathogen.  It screens everything from drugs to the AIDS virus in humans.  ELISA techniques have advantages of specificity and sensitivity when compared with more traditional tests.  It is generally a cheap and simple method.

        The ELISA is based on antibody recognition of a particular antigenic epitope.  Monoclonal antibodies can be cross-linked to one of nine different enzymes and used in the ELISA.  Anti-rabbit immunoglobulin is conjugated with alkaline phosphatase (1:3 by weight) by addition of glutaraldehyde.  Glutaraldehyde is a bifunctional cross-linker used to join the enzyme to the antigen or antibody.  Maleimide derivatives can link two separate protein molecules together, one through an amide bond and the other through a thioether bond.  Based on the principle of antibody-antibody interaction, the ELISA technique allows for easy visualization of results and can be completed without the additional concern of radioactive materials use.


        There are many variable techniques for ELISA, indirect, sandwich, and competitive are described below:

An indirect EIA, the method that was used for the experiment carried out, can be used to detect specific antibodies in an antibody mixture, for example. This technique can be used to check if an individual is infected with HIV or is exposed to tuberculosis by testing their serum for presence of antibodies. In this case the desired antigen is attached to the well surface, and is carried out as follows:

A specific primary antibody (or antibody mixture) is added to the wells.  If the antibody recognizes the antigen, Antibody-Antigen complexes form.  When the wells are rinsed, all unattached antibodies are removed from the well.  A secondary antibody is then added to the wells. This antibody only binds if a primary antibody is bound to the antigens in the well as it recognises the Fc portion of other (primary) antibodies.  The wells are rinsed again. If no primary Antibody - Antigen complexes are present, and then any secondary antibody that was added will be washed away.  The secondary antibody is conjugated to an enzyme that catalyses a chromogenic reaction, which results in a color reaction when the color substrate is added to the wells.  Therefore if there was no secondary antibody present, there will be no colour reaction. The diagram below shows the order of reagents for the process:

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Figure A

The Sandwich EIA technique allows for the detection of a specific antigen in a fluid sample.  For example, to detect hCG (a pregnancy hormone) in urine.

For this method the antigen samples are added to wells containing a specific antibody.  If the specific antigen is present in the sample, it will bind to the antibody coating.  The wells are then rinsed, removing any unbound antigens out of the wells.  Another antibody, identical to the first (except that it is conjugated to an enzyme) is now added to the wells.  The enzyme-linked antibody binds to any exposed antigenic sites, ...

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