Detection Of B. Anthracis Spores Using Monoclonal Antibodies

Bacillus anthracis, the causative agent of anthrax, is a rod-shaped, Gram-positive and spore-forming bacterium. In response to nutrient deprivation, B. anthracis will produce spores that can withstand harsh environments, and thus could be used as a biothreat reagent. Therefore, it is vital to develop a rapid, convenient method to detect B. anthracis.In the current study, three specific and high affinity monoclonal antibodies (mAbs), designated 8G3, 10C6 and 12F6, were obtained using inactivated B. anthracis spores as an immunogen. These mAbs not only can recognize the surface of B. anthracis spores, but also can detect intact vegetative cells. Furthermore, they had no cross-reaction with B. cereus,B. thuringensis and other relatives. Among these mAbs, mAb 8G3 had the highest affinity with B. anthracis spores, while mAb 12F6 seemed to be the best candidate for the detection of B. anthracis vegetative cells. The three mAbs were all determined to be directed against EA1 protein.Although EA1 is well known as a major surface layer (S-layer) component of B. anthracis vegetative cells, there were different opinions about the question of "Whether EA1 is also a true spore protein or not". Our results showed that, during the spore purification, this protein persistently existed on the spore surfaces with great amount. Besides this, the B. anthracis spores, even after rigous washing, can be detected by our mAbs. Therefore, whatever the answer is, EA1 could be used as a decection target of B. anthracis spores,In this study, we also firstly demonstrate the first use of surface plasmon resonance (SPR) biosensor for the rapid, sensitive and label-free detection of whole B. anthracis spores. The approach involved the use of a SPR biosensor (BIAcore 3000), a monoclonal antibody which was raised against B. anthracis spore (mAb 8G3). By means of subtractive inhibition assays, the detection limit of 10~4 CFU/ml is achieved within 40 min, and the other related Bacillus spores even with high concentration can be easily differentiated from B. anthracis spores by this approach. Therefore, the method could be used as an alternative detection means in central lab and potentially applied in field conditions when the portable SPR machine is available.