Detection of Frequency Resonance Energy Transfer Pair on Double-Labeled Microsphere and Bacillus anthracis Spores by Flow Cytometry

Abstract

Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare. Fluorescence methods combined with immunodiagnostic methods are the most common. To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method. FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET. Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method. This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system. As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores. The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B. anthracis exosoporium components. The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor. On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (E q ‫؍‬ 35%) and appearance of the alexa594 fluorescence (E s ‫؍‬ 22%), as detected by flow cytometry analysis. The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B. thuringiensis subsp. israelensis and B. subtilis. This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B. anthracis spores by a factor of 10 with respect to B. thuringiensis subsp. israelensis and a factor of 100 with respect to B. subtilis as control spores.Development of an ultrasensitive biosensor for biological hazardous in the environment is a major need for pollutant control and for the detection of biological warfare (16,22). The pathogen bacteria Bacillus anthracis is one of the biological agents likely to be used as a bioweapon (7); hence, it is the focus of a major effort in the field of rapid detection and identification (15). Fluorescence methods combined with immunodiagnostic methods are widely used for the detection and identification for biological hazards in the laboratory (8, 25, 28, 37; H. Kulaga, P.-E. Anderson, M. C. Cain, and P. J. Stopa, Proc. 1997 erdec Sci Conf. Chem. Biol. Defense Res. 1997) or the field (1, 30).Philips et al. have shown that B. anthracis spores can be identified by fluorescence microscopy and flow cytometry after appropriate labeling of the sample using fluorescent labeled antibodies (24,26,28). Nevertheless, difficulties in the selective identification of B. anthracis in mixed population samples were previously encountered (27). Recently, Stopa (37) has shown that ...

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

“…Preliminary characterization has shown that the antibody has no cross-reactivity with other phylogenetically related Bacillus spores (Fig. 3), demonstrating improved specificity compared to that of polyclonal antibodies generated in our group by means of the same exosporium fraction (27). As demonstrated in our previous work, implementation of the polyclonal antibodies for specific detection of B. anthracis was achieved solely by using a double-staining method (fluorescence resonance energy transfer), whereas the scFv antibody enabled direct specific detection.…”

“…This high-molecular-weight band is probably the Bcl1 protein, known as the most immunogenic antigen of the exosporium fraction (14,20,22). The affinity constant of the purified scFv antibody was determined via FCM as demonstrated previously (18,27). In short, mean spore fluorescence at steady state was measured as a function of the antibody concentration.…”

“…The same exosporium fraction was applied in the past for the successful preparation of polyclonal anti-B. anthracis spore antibodies in rabbits (27), resulting in highly reactive antibodies. The constructed library contained 7 ϫ 10 6 independent scFv recombinants, a population characteristic of immunized phage display libraries (9,24).…”

“…Analysis was performed using CellQuest Pro (Becton Dickinson) and FlowJo software. The FCM setup for spore analysis was described elsewhere (27). For specificity determination, different spores (B. anthracis, B. cereus 569, and B. subtilis DSM 675) were applied in the assay (5 ϫ 10 6 CFU/ml), with 1 nM scFv antibody.…”

“…The affinity of the purified scFv antibody for B. anthracis spores was determined using flow cytometry (FCM) on a FACSCalibur machine (Becton Dickinson Immuno Cytometry Systems, Mississauga, Ontario, Canada), as described previously (18,27). Each test tube contained B. anthracis spores (1 ϫ 10 6 CFU/ml) and anti-E-tag (Pharmacia) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse (F2266; Sigma-Aldrich, St. Louis, MO) (2 g/ml) antibodies.…”