An immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody to its antigen.
Both the presence of antigen or antibodies can be measured. For instance, when seeking to detect the presence of an infection the concentration of antibody specific to that particular pathogen is measured
The antibodies used may be polyclonal or monoclonal, each with characteristics fitting them for use in particular immunochemical methods.
Monoclonal antibodies are a single type of antibodies having the same antigenic determinant produced by a single hybridoma clone.The hybridoma is made by fusing a lymphocyte with a myeloma cell.presence of a single antigenic determinant is the useful feature of the monoclonal antibodies.they bind with only one type of epitope on the antigens.
Polyclonal antibodies (or antisera) are antibodies that are derived from different B cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.
The most important immunochemical methods include the following:
Immunolocalization is a technique for identifying the presence of a protein within is a technique for identifying the presence of a protein within the cell, its relative abundance and its subcellular localization. After suitable preparation of the cells, they are treated with an antibody (the primary antibody) that binds to the protein of interest. An antibody that binds to the primary antibody (the secondary antibody) is then allowed to bind and form an antigen—primary antibody—secondaryantibody complex.
Western blotting is a widely used technique in which antibodies are used to is a widely used technique in which antibodies are used to detect proteins following electrophoresis, generally SDS polyacrylamide gel electrophoresis that permits the separation of proteins on the basis of their molecularweights . Western blotting can be used to determine the presence and relative amount of a particular protein in a biological sample as well as its molecularweight.
Radioimmunoassay (RIA) is a very sensitive method used to measure minute quantities(RIA) is a very sensitive method used to measure minute quantities of an antigen. Since this method is most often used to measure drugs, toxicants,and other xenobiotics, the antigen used to produce the antibody is the small molecule(hapten) linked covalently to a protein.