The recent development of a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) immunoassay enables one-step, rapid (minutes), and direct detection compared to the multistep, time-consuming (hours), heterogeneous ELISA-type immunoassays. The use of the time-resolved effect of a donor lanthanide complex with a delay time of microseconds and large Stokes shift enables the separation of positive signals from the background autofluorescence of the sample. However, this study shows that the sample matrices directly interfere with donor fluorescence and that interference cannot be eliminated by time-resolved settings alone. Moreover, the reduction in donor emission did not appear to be equivalent to the reduction in acceptor emission, resulting in incorrect FRET signal measurements. To overcome this limitation, an internal standard approach was developed using an isotype control antibody. This new approach was used to develop TR-FRET assays for rapid detection (15-30 min) of Bacillus anthracis spores and botulinum toxin (type E) in beverages, which are major concerns in bioterrorism involving deliberate food contamination. Additionally, we demonstrate the detection of B. anthracis-secreted protective antigen (PA) and the Yersinia pestis-secreted markers F1 and LcrV in blood cultures, which are early markers of bacteremia in infected hosts following a possible bioterror attack. The use of an internal standard enables the calculation of correct ΔF values without the need for an external standard. Thus, the use of the internal standard approach in homogeneous immunoassays facilitates the examination of any sample regardless of its origin, and therefore expands the applicability of TR-FRET assays for complex matrices.
Keywords: Bacillus anthracis; Botulinum toxin; FRET; Time-resolved fluorescence; Yersinia pestis.