Novel immuno-FRET assay method for Bacillus spores and Escherichia coli O157 : H7

Novel immunofluorescence resonance energy transfer (immuno-FRET) assays for both Bacillus cereus spores and Escherichia coli O157:H7 are reported. Bot h assays involve the use of dual (QSY-7 and Oregon Green 514-antibody)-labe led spores or vegetative bacteria, such that Oregon Green 514-labeled antib odies are quenched by proximal QSY-7 molecules that are covalently bound to the dual (Oregon Green 514 and QSY-7)-labeled cells. Upon introduction of unlabeled bacteria or spores, in the respective assays, an increase in fluo rescence is observed in proportion to the numbers of unlabeled cells. This is due to migration of Oregon Green 514-labeled antibody from the dual-labe led cells to the unlabeled target cells as verified by fluorescence microsc opy. Optimization of the QSY-7 surface density led to a B. cereus spore det ection sensitivity of approximately 1.0 x 10(5) to 2.5 x 10(5) spores per m illiliter and 3.5 x 10(5) cells per milliliter for E. coli using a conventi onal cuvette-based spectrofluorometer. (C) 2001 Academic Press.