Using flow cytometry to develop a competitive assay for the detection of biotin

Abstract

BackgroundFlow cytometry (FCM) has many advantages including detection of bioparticles at low concentrations. A competitive assay for the detection of protein-binding compounds, such as biotin, was developed. MethodsUsing the fluorescein-biotin and streptavidin-display spores, biotin could be easily detected and quantified by FCM. The assay was made up of three steps: (1) mixing the sample and the spores containing streptavidin displayed on their surface, (2) mixing with fluorescein-biotin, and then adding biotin for competition, and (3) injecting the resultant mixture into a flow cytometer. Significant findingsWhen the sample-mixing time, the time required to mix the sample and recombinant spores, was ∼1 min and the total assay time was ∼10 min, the detection limit of proposed assay was ∼5 × 10-17 mol, which is 20-fold higher than that of ELISA. However, the detection limit was found be to as low as 3 × 10-18 mol when the mixing time was ∼18 hr. The proposed assay takes less assay time and better detection limit as compared to ELISA with the total assay time of ∼2-3 hr and the complex and tedious procedures. The developed FCM-based competitive assay can be applied in the detection of haptens (e.g., dioxin).