Avidin biotin binding ability

Use of avidin-biotin molecules in immunocytochemistry dramatically increases the detection sensitivity and greatly increases the flexibility of labels. The power of avidin-biotin is their ability to bind with extremely high affinity to each other. In addition, the ligand (biotin) and receptor (avidin) are unique for each other. There are multiple ways that labels can be conjugated to avidin or biotin and then used in immunocytochemistry. This section will introduce these molecules and show a several methods for their use in immunocytochemistry. 

A few years later egg-white injury in chicks was shown to be associated with a deficiency of biotin in the tissues, despite its abundance in the diet [145]. In the same year, avidin was isolated from egg-white and its ability to inactivate biotin in vitro was demonstrated [146]. Gyorgy and Rose [235] fed rats with avidin but only found very small amounts of biotin in the faeces until the faeces were steamed. Biotin was then released from the avidin-biotin complex. As the result of further experiments, it was concluded that the fundamental cause of egg-white injury is the unavailability of biotin due to its fixation to avidin, so that biotin is not absorbed from the intestinal tract and is excreted in the faeces [237]. A similar conclusion was reached by Sullivan and Nicholls [603] who showed that when egg-white is cooked, avidin is denatured and rendered incapable of binding biotin. Egg-white injury has been produced experimentally in man and can be cured by the administration of biotin [607]. In a recent study, Peters [497] reported that raw egg-white has a direct toxic effect which is not associated with its action in causing biotin deficiency. It would appear, therefore, that further studies on egg-white injury must be more closely associated with a critical analysis of the different components of egg-white. 

Biotin is widely distributed in foods, and deficiency is unknown, except among people maintained for many months on total parenteral nutrition and a very small number of people who eat abnormally large amounts of uncooked egg. There is a protein in egg white, avidin, that binds biotin extremely tightly and renders it unavailable for absorption. Avidin is denatured by cooking and then loses its ability to bind biotin. The amount of avidin in uncooked egg white is relatively small, and problems of biotin deficiency have occurred only in people eating abnormally large amounts — a dozen or more raw eggs a day - for some years. 

Other substances that exhibit specific binding may be used to separate the free and the bound fractions when attached to a solid phase the ability of staphylococcal protein A to bind to the FC fragment of certain isotypes of IgG can be utilized the strong binding of the vitamin biotin to tetravalent avidin may also be employed. Biotin may be readily incorporated into antibody molecules and these molecules may be subsequently captured by an avidin solid phase. Alternatively avidin may be used to provide a link between a biotinylated antibody and a biotinylated solid phase. 

Streptavidin may be immobilized to a surface using methods used with other proteins. For instance, strept(avidin) can be adsorbed onto a number of surfaces and still retain the ability to bind biotin and biotinylated molecules. Strept(avidin)-coated magnetic beads, plastic combs, and silicon chips used in many applications are made in this manner. Alternatively, streptavidin may be covalently bound to form an oriented monolayer using a cysteine derivative [101]. 

The determination of the quantity of protein bound to the insoluble carrier sometimes causes difficulties. The methods usually applied are laborious or somewhat inaccurate. Labeling of assayed protein, for instance with C-acet-anhydride, makes it possible to carry out a very fast and exact determination of immobilized protein The determination of bound enzyme C-labeled aldolase after its immobilization on polyacrylamide can serve as an example The concentration measurements of certain proteins are based on their ability to bind certain ligands. Radiolabels such as or H-biotin have been used for the determination of avidin by direct binding or for biotin assay by isotopic dilution Cofactor and fluorescent labeled ligands have been also used for the monitoring of specific protein binding reactions. 

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