Development of Antibodies against Anthrose Tetrasaccharide for Specific Detection of Bacillus anthracis Spores

Abstract

Methods for the immunological detection ofThe BclA protein is a major component of the exosporium of B. anthracis spores and is decorated by the tetrasaccharide indicated above. 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. The 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, such as enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and electron microscopy, revealed the specificities 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 showed only minimal cross-reactivities with other Bacillus strains. Moreover, the antibodies generated proved functional in a new capture assay for B. anthracis spores and could therefore be useful for the detection of spores in complex samples.

Identification of an African Bacillus anthracis Lineage That Lacks Expression of the Spore Surface-Associated Anthrose-Containing Oligosaccharide

“…Conjugate vaccines were produced by linking synthetic anthrose-containing saccharides to protein carriers. Immunization of animals with these vaccines allowed the production of monoclonal antibodies (MAbs) and polyclonal antibodies that cross-reacted with native B. anthracis spores (5,11,(26)(27)(28), confirming the structural analysis of the oligosaccharide and its expression on the endospore surface. Screening of antitetrasaccharide MAbs and anti-anthrose-rhamnose disaccharide MAbs for cross-reactivity with spores of a broad spectrum of B. anthracis strains and related species of the Bacillus genus demonstrated a broader presence of anthrose in the B. cereus group than initially expected (26,27).…”

Antibody Responses to a Spore Carbohydrate Antigen as a Marker of Nonfatal Inhalation Anthrax in Rhesus Macaques

“…The structure of this oligosaccharide is a ␤-Ant- (3,6,20,21,33,37). Rabbits vaccinated with live or irradiated killed B. anthracis Sterne spores mounted an immune response against a synthetic anthrosyl-containing trisaccharide (ATS) (Fig.…”

Fast and Sensitive Detection of Bacillus anthracis Spores by Immunoassay

“…Using antibodies directed against the anthrose tetrasaccharide, Kuehn et al developed a specific and sensitive ELISA able to detect 1 ϫ 10 4 to 5 ϫ 10 4 inactivated B. anthracis CDC1014 spores/ml (16). The most sensitive spore detection performed by means of a two-site immunometric assay was achieved using Luminex technology with a detection limit of 10 3 to 10 4 spores per ml (31).…”

“…Specific immunodetection of B. anthracis spores is challenging because of possible cross-reactivity of the antibodies (Abs) with near-neighbor species such as Bacillus cereus and Bacillus thuringiensis. Antibodies against the exosporium (28), the vegetative cells (19), oligosaccharide epitopes of the BclA spore surface protein (16,32,33), and extracellular antigen I (EAI) have been raised and used in immunoassays for B. anthracis detection (35). B. anthracis spores were successfully detected by immunofluorescence and cytometry techniques (20,21,27), but not with high specificity, because polyclonal antibodies were used in both cases and these methods are not suitable for samples containing a small quantity of target spores overwhelmed by other organisms in a complex matrix such as soil.…”