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Anti-Carbohydrate Antibodies for the Detection of Anthrax Spores.

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Angewandte
Chemie
Pathogen Detection
DOI: 10.1002/anie.200602048
Anti-Carbohydrate Antibodies for the Detection
of Anthrax Spores**
Marco Tamborrini, Daniel B. Werz, Joachim Frey,
Gerd Pluschke, and Peter H. Seeberger*
Spores of Bacillus anthracis have been used as biowarfare
agents to terrorize civilian populations. Once this durable
form of the pathogen has been inhaled it will kill most victims
if treatment does not commence within 24–48 h.[1] After
uptake into the body, the spores germinate and vegetative
cells multiply and produce toxins, leading to the development
of lesions from which the infection may disseminate, which is
frequently fatal. It is assumed that an effective anthraxsubunit vaccine should contain multiple antigens. A surface
antigen of the spores eliciting antibodies that suppress
germination and enhance phagocytosis of spores by macrophages would be a suitable component for such a vaccine.
Several detection systems have been developed to identify
B. anthracis[2–4] and process the large number of samples that
have to be tested each year. PCR-based assays or traditional
phenotyping of bacteria are the most accurate but are also
complex, expensive, and slow. Antibody binding to sporesurface peptide and protein antigens is less complicated,
although it is plagued by higher false-positive and falsenegative readouts. The similarity of spore-cell-surface antigens of the opportunistic human pathogen B. cereus and other
bacteria of this group makes it difficult to create selective,
reliable, antibody-based detection systems.
On the surface of B. anthracis spores, a unique tetrasaccharide 1 (Scheme 1) containing an entirely novel monosaccharide, named anthrose, was discovered recently.[5]
Access to usable quantities of pure cell surface oligosaccha-
[*] Dr. D. B. Werz, Prof. Dr. P. H. Seeberger
Laboratory for Organic Chemistry
Swiss Federal Institute of Technology (ETH) Z=rich
ETH H>nggerberg, HCI F 315
Wolfgang-Pauli-Str. 10, 8093 Z=rich (Switzerland)
Fax: (+ 41) 44-633-1235
E-mail: seeberger@org.chem.ethz.ch
M. Tamborrini, Prof. Dr. G. Pluschke
Swiss Tropical Institute
Socinstr. 57, 4002 Basel (Switzerland)
Prof. Dr. J. Frey
Institute of Veterinary Bacteriology
University of Bern
LInggassstr. 122
3012 Bern (Switzerland)
[**] This research was supported by ETH Z=rich, by a Feodor Lynen
Research Fellowship of the Alexander von Humboldt Foundation,
and by an Emmy Noether Fellowship of the Deutsche Forschungsgemeinschaft (to D.B.W.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2006, 45, 6581 –6582
Scheme 1. Structure of the tetrasaccharide 1 found on the surface of
B. anthracis spores and its synthetic analogue 2. Attachment through a
pentenyl handle to the KLH carrier protein afforded the conjugate 3:
a) O3, MeOH, 78 8C, 10 min; b) Me2S, MeOH, RT, 2 h; c) KLH carrier
protein, PBS buffer solution (pH 7.2), NaBH3CN, 37 8C, 48 h. PBS = phosphate-buffered saline.
rides by isolation is often difficult and leaves synthetic
chemistry as a last resort to procure carbohydrate antigens.
We envisioned the detection of B. anthracis spores based on
antibodies against carbohydrate antigen 1, which is not
present on related strains of bacteria.
The tetrasaccharide was chemically synthesized[6, 7] in the
form of the n-pentenyl glycoside 2.[6] The chemical handle on
the reducing end was utilized to introduce a reactive terminal
aldehyde moiety through ozonolysis. Covalent attachment[8, 9]
of synthetic 2 to the keyhole-limpet-hemocyanine (KLH)
carrier protein by reductive amination produced a carbohydrate–protein conjugate 3 that was immunogenic in mice
(Scheme 1). The conjugation to protein carriers is an efficient
way to improve the immunogenicity of oligosaccharide
antigens. The proteins provide peptide units that can be
recognized by carrier-specific T cells, thus converting a T-cellindependent response against an unconjugated oligosaccharide into a more efficient T-cell-dependent response.
Tetrasaccharide–KLH conjugates formulated in ImmunEasy adjuvant (QIAGEN) elicited tetrasaccharide binding
immunoglobulin G (IgG) antibodies after the third immunization in mice. Three B cell hybridoma lines producing
tetrasaccharide specific monoclonal IgG antibodies (mAbs)
were generated from spleen cells of one of the immunized
mice. In an indirect immunofluorescence assay, all three
mAbs bound specifically to native B. anthracis endospores.
No binding was found to endospores of B. cereus, B. subtilis,
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6581
Communications
[3] A. Fasanella, S. Losito, R. Adone, F. Ciuchini, T. Trotta, S. A.
Altamura, D. Chiocco, G. Ippolito, J. Clin. Microbiol. 2003, 41,
896 – 899.
[4] X. Zhang, M. A. Young, O. Lyandres, R. P. van Duyne, J. Am.
Chem. Soc. 2005, 127, 4484 – 4489.
[5] J. M. Daubenspeck, H. Zeng, P. Chen, S. Dong, C. T. Steichen,
N. R. Krishna, D. G. Pritchard, C. L. Turnbough, Jr., J. Biol.
Chem. 2004, 279, 30 945 – 30 953.
[6] D. B. Werz, P. H. Seeberger, Angew. Chem. 2005, 117, 6474 – 6476;
Angew. Chem. Int. Ed. 2005, 44, 4315 – 4318.
[7] a) R. Adamo, R. Saksena, P. KovHč, Carbohydr. Res. 2005, 340,
2579 – 2582; b) R. Adamo, R. Saksena, P. KovHč, Helv. Chim. Acta
2006, 89, 1075 – 1089.
[8] G. Raguputhi, R. R. Koganty, D. Qiu, K. O. Lloyd, P. O. Livingston, Glycoconjugate J. 1998, 15, 217 – 221.
[9] Z.-G. Wang, L. J. Williams, X.-F. Zhang, A. Zatorski, V. Kudryashov. G. Ragupathi, M. Spassova, W. Bornmann, S. F. Slovin, H. I.
Scher, P. O. Livingston, K. O. Lloyd, S. J. Danishefsky, Proc. Natl.
Acad. Sci. USA 2000, 97, 2719 – 2724.
Figure 1. Indirect immunofluorescent staining of B. anthracis and B.
cereus endospores by a mAb that was generated against the synthetic
tetrasaccharide 2. Left column: bright-field microscopy; right column:
Cy3-specific immunofluorescence staining of a) B. anthracis spores
with an isotype-matched control mAb, b) B. anthracis spores with a 2specific mAb, and c) B. cereus spores with a 2-specific mAb.
and B. thuringiensis, which are close relatives of B. anthracis
(Figure 1).
These results demonstrate that distinct differences in cellsurface carbohydrate antigens can provide the basis for the
generation of specific immunological reagents. This was
demonstrated herein for the synthetic tetrasaccharide antigen
2 of B. anthracis. The tetrasaccharide-specific mAbs may be
suitable to develop a highly sensitive and specific detection
system for B. anthracis endospores and contribute to the
development of novel therapeutic or preventive approaches.
Received: May 23, 2006
Published online: August 17, 2006
.
Keywords: anthrax · carbohydrates · monoclonal antibodies ·
oligosaccharides · pathogen detection
[1] R. M. Atlas, Annu. Rev. Microbiol. 2002, 56, 167 – 185.
[2] D. King, V. Luna, A. Cannons, J. Cattani, P. Amuso, J. Clin.
Microbiol. 2003, 41, 3454 – 3455.
6582
www.angewandte.org
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 6581 –6582
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