Enzymes are widely used in bioconjugate chemistry as detection components in assay systems. The catalytic activity of an enzyme can be used to turn substrate molecules into chro- mogenic, fluorescent, or chemiluminescent products, which are easily detectable or quantifiable by imaging, microscopy, or spectroscopy. If an enzyme is conjugated to a targeting molecule specific for some analyte of interest, then an assay system can be constructed to localize or measure the analyte. The most common targeting molecule is an antibody having antigen-binding specificity for the substance to be measured. An enzyme conjugated to such an anti-body can be used to visualize the presence of antigen. Due to the advantages of this simple concept, enzyme linked immunoadsorbent assays (ELISAs) have become the most important type of immunoassay system available. Besides, the flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques.
Biotinylated enzymes can be used as detection reagents in (strept) avidin–biotin assay procedures. Particularly, in the bridged avidin–biotin (BRAB) approach or the ABC technique, a biotin-labeled enzyme is used as the signaling agent after the binding to an antigen of a biotinylated antibody and an (strept) avidin bridging molecule. The biotins on the surface of the enzyme can bind with extraordinary affinity to (strept) avidin–antibody complexes, providing near-covalent interaction potential with high specificity.
There are some principal enzymes used for conjugation with other protein molecules, particularly in the design of ELISA and other immunoassay systems, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase. Adding a biotin label to an enzyme molecule is simple, given the wide variety of options available. Enzymes may be modified to contain reactive groups useful for conjugation with other proteins. This operation may be done using homobifunctional or heterobifunctional reagents that can covalently couple to some chemical target on the enzyme and result in a terminal reactive group that can crosslink with another molecule. Enzyme activation also may take advantage of the presence of polysaccharide constituents-oxidizing them with sodium periodate to form reactive aldehydes. Whatever the method of conjugate creation, the most important considerations are the retention of activity in the complex and prevention of extensive oligomer generation, which may cause precipitation. The more common methods for producing enzyme conjugates include:
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