Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays.

We have achieved sensitive, rapid and reproducible detection of three biological threat agents in a variety of biological and environmental matrices using the DELFIA time-resolved fluorometry (TRF) assay system (Perkin-Elmer Life Sciences, Akron, OH). Existing ELISA assays for the detection of Francisella tularensis, Clostridium botulinum A/B neurotoxin (BotNT A/B), and Staphylococcus aureus enterotoxin B (SEB) were converted to TRF assays. They use 100 microl of positive control or unknown per test well and require just over 2 h to run. Fluorescent signal read time is a fraction of a second per well. The assay format consists of a capture ELISA utilizing a biotinylated capture antibody, prebound to a streptavidin-coated 96-well plate and a lanthanide (Europium, Eu3+)-labeled detector antibody. The bound Eu-labeled detector antibody produces a fluorescent signal upon the addition of an enhancement solution. The signal results from the dissociation of the Europium from the antibody, creating a micelle, thus amplifying the signal nearly one million-fold. Sensitivities achieved by these assays were between 4 and 20 pg/ml in buffer. Additionally, we have tested this system in different matrices such as serum, urine, dirt, and sewage. Concentration curves generated from standard solutions produced a wide linear range making serial dilutions of unknown samples unnecessary. DELFIA TRF assays are significantly better in terms of sensitivity, linear range, and run time than standard capture ELISAs and should facilitate early detection of potential biological warfare agents in clinical and environmental samples.