CVI Accepts, published online ahead of print on 30 September 2009

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Clin. Vaccine Immunol. doi:10.1128/CVI.00235-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development of antibodies against anthrose tetrasaccharide for specific detection of B. anthracis spores

Andrea Kuehn, Pavol Kovác, Rina Saksena, Norbert Bannert, Silke R. Klee, Heidrun Ranisch, and Roland Grunow^*

Robert Koch-Institut, Centre for Biological Security 2, Nordufer 20, D-13353 Berlin, Germany; Robert Koch-Institut, Centre for Biological Security 4, Nordufer 20, D-13353 Berlin, Germany; Section on Carbohydrates, National Institutes of Health, NIDDK, Laboratory of Bioorganic Chemistry, 8 Center Drive, Bethesda, MD 20892-0815, USA

^* To whom correspondence should be addressed. Email: GrunowR{at}rki.de.

The immunological detection of Bacillus anthracis in various environmental samples and the discrimination from other members of the B. cereus group is not well established yet. To generate specific discriminating antibodies, we immunized rabbits, mice and chicken with inactivated B. anthracis spores and, additionally, rabbits and mice with the tetrasaccharide [–Ant–(13)––L–Rhap–(13)––L–Rhap–(12)–L–Rhap]. It is a constituent of the exosporium glycoprotein BclA and contains the newly discovered sugar anthrose [2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy--D-glucose]. The BclA protein is a major component of the exosporium of B. anthracis spores and is decorated by the above tetrasaccharide. The anthrose-containing tetrasaccharide chain seems to be highly specific for B. anthracis, which makes it a key biomarker for the detection of these spores. Different immunizations led to anthrose-reactive polyclonal and monoclonal antibodies which were analyzed by various methods to characterize their ability to discriminate between B. anthracis and other Bacillus spp. Multiple applications like ELISA, indirect immunofluorescence assay and electron microscopy revealed specificity of the polyclonal and monoclonal antibodies generated for B. anthracis spore detection. All polyclonal antibodies were able to correctly identify the B. anthracis strains tested and only showed minimal cross-reactivities with other Bacillus strains. Moreover, the generated antibodies proved functional in a new capture assay for B. anthracis spores and could, therefore, be useful for the detection of spores in complex samples.