For each screening of the hybridoma cultures, spores from B. cereus and B. thuringiensis, the two closest relatives of B. anthracis, were used as negative controls. To identify mAbs with high affinity and specificity, hybridomas were selected if the mAbs reacted strongly with B. anthracis but did not recognize either negative antigen. As a result,GSK2118436 the three mAbs we prepared have no cross reaction with many B. thuringienesis subspecies and B. cereus isolates. The three mAbs recognized not only the surface of B. anthracis spores but could also detect intact B. anthracis vegetative cells. Furthermore, these mAbs were capable of reaction with live B. anthracis as well as dead B. anthracis, which is critical for the detection of biological warfare agents in unknown ‘‘white powders’’, since it has been suggested that Bacillus inactivation would affect antibody detection assays. Although these three mAbs were directed toward the same target protein, EA1, they had different characteristics. The mAb 12F6 was superior at reacting with different kinds of B. anthracis vegetative cells, while 8G3 had a higher affinity for B. anthracis spores and the target protein EA1. Besides this, in the epitope mapping, the epitopes of mAb 8G3 and 10C6 were concluded to be located from the amino acid 275 to 435 on the EA1 protein, and the epitope of mAb 12F6 was exactly located from the amino acid 465 to 554. We suggested that the different positions of the mAb epitopes caused the mAbs to exhibit different behavior the detection of B. anthracis. As to whether EA1, a major S-layer component of B. anthracis vegetative cells, is also a spore protein,GSI-IX much research has indicated that this protein is retained in the proteomic profiling of spores and salt/detergent washed exosporium. However, Williams and Turnbough stated that this protein was merely a persistent contaminant in spore preparations. Whichever is correct, it does not matter for the detection of B. anthracis spores, because this protein does persistently exist in each of the spore preparations, and the B. anthracis spores, even after full washing, can be detected with our anti-EA1 mAbs.