Colorimetric methods biotin

Leary, J.J., Waldrop, A.A., and Ward, D.C. (1983) Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose Bio-blots. Proc. Natl. Acad. Sci. USA 80, 4045 1049. 

As recently as 2008, a reliable colorimetric method has been reported for biotin in pharmaceuticals based on its catalytic impact on the azide-triiodide reaction (Walash et al. 2008). However, biotin is best chromatographically separated from other compounds to allow practical determinations in all but the simplest matrices. This is particularly true for biological and food materials. Reviews such as Livaniou et al. (2000) summarize many of the historical physico-chemical techniques. 

The natural substrate of biotinidase is biocytin, but it also readily cleaves an amine bond between biotin and a variety of different compounds, allowing the use of artificial substrates for the quantitation of biotinidase activity. The most common method is the colorimetric assay described by Knappe and coworkers in 1963 [14, 32], which uses biotinyl-p-aminobenzoic acid (B-PABA) as substrate. The assay of plasma biotinidase activity by this method allows rapid detection of individuals with biotinidase deficiency. 

In keeping with current trends in immunohistochemistry to develop alternatives to biotin-streptavidin detection methods, a fluorescyl-tyramide amplification system has recently been introduced (FT-CSA). In this procedure peroxidase is associated with a tissue-bound primary antibody by application of a secondary antimouse Ig antibody to which peroxidase has been conjugated. The peroxidase catalyzes the conversion and deposition of fluorescyl-tyramide onto the tissue section. At this point the reaction can be terminated and viewed by fluorescence microscopy, or the signal can be converted to a colorimetric reaction by the sequential application of an anti-fluorsecein antibody conjugated to peroxidase followed by a diaminobenzidine-hydrogen peroxide substrate. 

DB McCormick, JA Roth. Colorimetric determination of biotin and analogs. Methods Enzymol 18A 383-385, 1970. 

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