Identification of B. anthracis ATCC14185 spores in environmental samples. Environment samples collected from asphalt-paved roads, sidewalks, and various soils were spiked with 5 × 103–1 × 104 CFU/mL B. anthracis 14185 spores per sample. Samples were processed and resuspended in 400 µl DMEM supplemented with 50% FBS and incubated at 37 °C in 10% CO2 for 5 h. Following incubation (toxins biosynthesis process), supernatants from each sample were analyzed for secreted EF, LF, and PA in the new multiplexed immune assay. (A) Detection of EF/LF/PA was assessed following the incubation process of environmental samples (n = 10) spiked with 5 × 103 CFU B. anthracis spores, negative environmental samples (n = 10), and 5 × 103 CFU spores suspended in growth medium as a control. (B) Detection of EF/LF/PA was assessed, followed by an incubation process of 40 different environmental samples collected from four different geographic locations in Israel (marked as A-D) spiked with 1 × 104 CFU B. anthracis spores. Results are presented as averages ± SE (error bars) of signal to noise ratios (S/N). Positive detection was defined as S/N ≥ 2 (black dashed line).