As to whether EA1, a major S-layer component of B. anthracis vegetative cells, is also a spore protein, 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. Although EA1 could be partially washed out during the rigorous washing step in our study, the other proteins detected by SDSPAGE also BLZ945 apparently decreased and were present as debris in the supernatant of centrifuged purified samples. The supernatant debris contained significant amounts of true spore proteins with 10-Undecenoic acid similar protein profiles to fully washed spores. Therefore, rigorous washing methods, such as Renografin purification, can cause serious loss of spore surface associated proteins and are not suggested for proteomic analysis. This suggests that EA1 is certainly at least a highly spore-associated protein: it might be a true spore protein, or may anchor onto particular spore surface components. In conclusion, this study reports three mAbs that can bind to B. anthracis spores and intact vegetative cells with high species-specificity and affinity. It also indicates that EA1, the target protein of our mAbs, could serve as a potential detection target of B. anthracis, establishing a new immunoassay protocol that realizes sensitive, rapid, on-site and simultaneous detection of both life forms of B. anthracis. Nestin is an intermediate filament protein which exhibits structural similarities to vimentin, desmin and neuro-filaments and is classified as a type IV neuro-filament. Nestin is assembled into intermediate filaments by forming heterodimers with vimentin and desmin and together with microtubules and microfilaments it forms the cytoskeleton.