TUTORIAL ON ELISA( Enzyme-linked Immunosorbent Assay)
Enzyme-Linked Immunosorbent Assay
An antibody (Ab) reacts with an antigen (Ag) in a highly specific manner. This means that an antibody reacts only with that determinant or region of an antigen for which it is specific. To form 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.
In either case, either the amount of Ag-Ab complex formed or the rate of its formation is used to determine the quantity of either the antigen or the antibody involved in the interaction.
The various assays used for the purpose are as follows:
1. Precipitin reaction
2.Ouchterlony assay
3. Mancini assay
4. Immunoelectrophoresis
5. Western blotting
6. Rocket electrophoresis
7. Agglutination reactions
8. Labelled antibody techniques
9. Direct or primary method
10.Indirect labelled
Under indirect labelled antibody techniques come
i) Radioimmunoassay (RIA)
ii) Enzyme-linked immunosorbent assay (ELISA)
iii) Fluorescent labels
PROCEDURE OF ELISA
A generalized procedure and the basic principle of ELISA is as follows.
The antigen of interest is immobilized on the surface of a test tube, petriplate or microtitre well. Now, antibody specific to antigen is added and allowed to react with absorbed antigen. Unreacted molecules of antibody are washed away leving only Ag-Ab complex. The antigen-specific antibody is normal or unlabelled. This constitutes the primary reaction.
In the secondary reaction, the anti-immunoglobulin ( an antibody that reacts with all the antibodies belonging to the same class) is added into the vessel and allowed to react with tha 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 (i.e. is labelled with an enzyme molecule) in such a way that the anti-Ig activity is not impaired. The unreacted anti-Ig is washed away and finally the substrate of the enzyme is added along with the necessary reagents to develop colour due to enzyme activity. The intensity of colour development is proportional to the quantity of antigen/antibody. For an easy and rapid assay, a computerised ELISA reader may be used. The sensitivity of ELISA is in the range of nanograms. This method of ELISA is often called direct antigen coating (DAC) ELISA. This approach is useful in estimating the amount of specific antibody present in the anti-serum used for the primary reaction.
In another approach, an unlabelled antibody specific to the antigen of interest is immobilized in a microtitre well. The antigen is added and allowed to react with the immobilized antibody to form Ag-Ab complex. A second antibody specific to the antigen, and labelled with an enzyme is now added, which reacts with the Ag-Ab complex. The unreacted antibodies are washed out and enzyme substrate is added. The colour change is measured. It is essential for this approach that the antigen has atleast two accessible binding sites for antibodies. This approach is used for the detection or qualitative assay of the antigen of interest and is called double antibody sandwich (DAS) ELISA.
The antibody immobilization may be achieved by using the wall protein A of Staphylococcus aureus. Protein A binds non-specifically to all entibodies in their Fc portion (crystal forming fragment) . the protein A may be first immobilized in the microtitre well, to which the unlabelled antigen-specific antibody is added. The antibody becomes immobilized due to the binding of Protein A in the Fc region.
The enzymes used for labelling of antibodies are horseradish peroxidase, alkaline phosphatase, beta-galactosidase, lactoperoxidase etc.
APPLICATIONS OF ELISA
Useful for determining Serum antibody concentrations in
1. HIV AIDS test
2. West Nile Virus test
3. Food industry(detecting potential food allergens such as milk, peanuts, walnuts, almonds and eggs)
4. Detection of proteins and hormones in human body
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