29] Fluorophore-linked assays for high-performance liquid chromatography postcolumn reaction detection of biotin and biocytin

Abstract

Publisher Summary This chapter describes high-performance liquid chromatography (HPLC) postcolumn reaction detection systems in which two types of protein-binding assay principles are used. In a competitive-binding approach, the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) competes with the analyte (biotin or biocytin) for the same binding sites on avidin. Two noncompetitive binding approaches are also described, in which the interaction that takes place is between the analyte (biotin or biocytin) and avidin or streptavidin. In these cases (strept)avidin is labeled with fluorescein isothiocyanate (FITC). On binding with the analyte, the fluorescence intensity of (strept)avidin–FITC increases in a manner proportional to the concentration of the analyte. The coupling of the binding assays with HPLC results in a highly selective detection of biotin and its derivatives owing to their selective interaction with (strept)avidin. These types of approaches also lead to enhanced sensitivity because the HPLC detection is fluorescence based rather than the conventional ultraviolet (UV) absorbance-based detection. The chapter also describes an absorbance-based postcolumn reaction detection system. In this system, biotin competes with and displaces the dye 2-(4'-hydroxyphenylazo)benzoic acid (HABA), where the free and bound HABA have different absorbance characteristics.