Development of an N-hydroxysuccinimidyl fluorescein-O-acetate-labeled probe for competitive capillary electrophoretic immunoassay of bovine serum albumin

A highly fluorescent reagent, N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) has been labeled on bovine serum albumin (BSA) to construct a new immunofluorescent probe, SIFA-BSA, for the competitive immunoassay of BSA with capillary electrophoresis. Labeling conditions of SIFA with BSA such as the reaction molar ratio and reaction time, as well as separation conditions for free and antibody-bound SIFA-BSA including the pH and concentration of buffer were systematically investigated. Under the optimized conditions, SIFA-labeled BSA and free BSA competitively reacted with a limited amount of anti-BSA antibody. Free and antibody-bound SIFA-BSA could be well separated within 5 min using 100 mM boric acid buffer (pH 9.3) for background electrolyte, 28 kV for the separation voltage, and 25 °C for the column temperature. With the proposed method, a much lower detection limit (S/N = 3) of 0.1 μg/mL was obtained compared with the competitive capillary electrophoresis immunoassay of BSA with fluorescein isothiocyanate (FITC) labeled BSA probe, in which the detection limit (S/N = 3) is 0.0031 mg/mL.