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Applied and Environmental Microbiology, April 2003, p. 2330-2339, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2330-2339.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

E. Zahavy,1* M. Fisher,2 A. Bromberg,1 and U. Olshevsky2

Departments of Physical Chemistry,1 Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona 74100, Israel2

Received 23 August 2002/ Accepted 26 December 2002

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 (Eq = 35%) and appearance of the alexa594 fluorescence (Es = 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.

* Corresponding author. Mailing address: Department of Physical Chemistry, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona 74100, Israel. Phone: 972-8-9381457. Fax: 972-8-9381743. E-mail: eran{at}iibr.gov.il.

Applied and Environmental Microbiology, April 2003, p. 2330-2339, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2330-2339.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mechaly, A., Zahavy, E., Fisher, M. (2008). Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores. Appl. Environ. Microbiol. 74: 818-822 [Abstract] [Full Text]  
  • Pai, S., Ellington, A. D., Levy, M. (2005). Proximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores. Nucleic Acids Res 33: e162-e162 [Abstract] [Full Text]  
  • Brigati, J., Williams, D. D., Sorokulova, I. B., Nanduri, V., Chen, I-H., Turnbough, C. L. Jr, Petrenko, V. A. (2004). Diagnostic Probes for Bacillus anthracis Spores Selected from a Landscape Phage Library. Clin. Chem. 50: 1899-1906 [Abstract] [Full Text]  
  • Yitzhaki, S., Barnea, A., Keysary, A., Zahavy, E. (2004). New Approach for Serological Testing for Leptospirosis by Using Detection of Leptospira Agglutination by Flow Cytometry Light Scatter Analysis. J. Clin. Microbiol. 42: 1680-1685 [Abstract] [Full Text]  


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