A homogeneous assay for biotin based on chemiluminescence energy transfer

Abstract

Chemiluminescence energy transfer between aminobutylethylisoluminol (ABEI)-biotin and fluorescein-avidin was investigated in order to establish a homogeneous assay for serum biotin in the physiological range. ABEI chemiluminescence was measured at pH 7.4 using microperoxidase-hydrogen peroxide and the chemiluminescence at two wavelengths (460 and 525 nm) measured simultaneously to quantify chemiluminescence energy transfer. ABEI-biotin was synthesized by a mixed anhydride reaction and purified by TLC and HPLC. Binding of ABEI-biotin to fluorescein-avidin resulted in a quenching of the chemiluminescence. Chemiluminescence energy transfer was demonstrated by a 2.5-fold decrease in the ratio of blue (460 nm) to green (525 nm) light emission compared with unbound ABEI-biotin. This energy transfer was used to establish an assay for biotin in the range 1 to 10 n m by relating the concentration of biotin to the ratio of chemiluminescence monitored at 460 and 525 nm simultaneously. The assay was capable of detecting biotin in reference sera and in patients with malabsorption syndromes and chronic alcoholism. The reference range in normal subjects was 1.2 to 4.3 nmol/liter mean ± SD = 2.41 ± 0.91 nmol/liter ( n = 20). The quenching of the chemiluminescence of ABEI-biotin when bound to fluorescein-avidin appeared to be the result of a direct interaction between the excited state product of ABEI and fluorescein.