Corbett Cindi R, Ballegeer Erin, Weedmark Kelly A, Elias M D, Al-Saleem Fetweh H, Ancharski Denise M, Simpson Lance L, Berry Jody D
National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Mannitoba, Canada.
Hybridoma (Larchmt). 2011 Dec;30(6):503-10. doi: 10.1089/hyb.2011.0032.
Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (H(C)50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H(C)50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of H(C)50/A (H(CN)25/A) comprising amino acid residues (985)WTLQDTQEIKQRVVF(999), an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis.
肉毒杆菌神经毒素（BoNTs）是极具毒性的毒素，可污染食物，是公共卫生关注的问题。已有能够检测BoNTs的抗BoNT抗体的报道；然而，对于BoNTs仍需要准确且灵敏的检测能力。在此，我们描述了一组针对使用高通量筛选方法开发的BoNT/A无毒受体结合域（H(C)50/A）产生的八种单克隆抗体（MAb）的特性。在两次独立的杂交瘤融合中，针对重组H(C)50/A产生了两组各四种IgG MAb。在这八种抗体中，只有一种单克隆抗体F90G5-3与完整的BoNT/A蛋白结合，并对其进行了进一步表征。F90G5-3单克隆抗体在体内小鼠生物测定中略微延长了死亡时间，并通过肽扫描定位到H(C)50/A（H(CN)25/A）N端亚结构域中的一个肽表位，该表位由氨基酸残基（985）WTLQDTQEIKQRVVF（999）组成，是一个在人类中具有高度免疫反应性的表位。此外，我们证明F90G5-3以纳摩尔效率结合BoNT/A。总之，我们的结果表明F90G5-3作为用于BoNT/A捕获测定开发和生物法医分析的诊断免疫试剂具有潜在价值。
