Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies

In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs.In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.Botulism is a disease which can be fatal if untreated and is caused by exposure to any one of the highly toxic protein family known as botulinum neurotoxins (BoNTs). In vivo, BoNT cleaves proteins necessary for nerve signal transmission. This enzymatic cleavage results in the inhibition of the nerve impulse, leading to flaccid paralysis of the victim which can affect the lungs and may necessitate ventilator support. Treatment of the botulism patient involves administration of therapeutic immunoglobulin and is most effective when administered within 24 h of toxin exposure [1]. Due to the extreme toxicity, global availability, and ease of preparation of BoNT, it is considered a likely agent for bioterrorism [2].Previously, our laboratory reported in several publications on the development of the Endopep-MS method as an assay for BoNT detection and serotype differentiation [3,4]. This method can detect all seven known BoNT serotypes and involves incubating BoNT with a peptide substrate that mimics each toxin's natural in vivo neuronal protein target. The presence of a particular BoNT serotype is demonstrated