Bioluminescent Homogeneous Enzyme Binding Assay for Biotin Using Luciferase as a Label

Abstract

Abstract Bioluminescent homogeneous enzyme binding assay was developed for biotin using recombinant firefly luciferase as a label. Biotinylated luciferase was prepared by the reaction of luciferase with biotinyl-e-aminocaproic acid N-hydroxyl succinimide ester. The biotinylated luciferase was inactivated by adding avidin. However, the inactivation was prevented in the presence of an excess amount of biotin. The measurable range of biotin was 15 pg/assay tube to 500 pg/assay tube. The detection limit of biotin was 15 pg/assay tube. The coefficient of variation (CV, n=5) for each point ranged from 1.4% to 11.9% (mean 7.9%). The proposed method is very convenient, rapid and sensitive by using homogeneous enzyme binding assay system and bioluminescent reaction as detection system.