Slagboom Julien, Lewis Abigail H, Schouten Wietse M, van Haperen Rien, Veltman Mieke, Bittenbinder Mátyás A, Vonk Freek J, Casewell Nicholas R, Grosveld Frank, Drabek Dubravka, Kool Jeroen
Amsterdam Institute of Molecular and Life Sciences, Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1085, Amsterdam, 1081HV, the Netherlands.
Department of Cell Biology and Genetics, Faculty of Medicine, Erasmus Medical Center Rotterdam, 3000 DR, Rotterdam, the Netherlands.
Toxicon X. 2024 Feb 16;21:100185. doi: 10.1016/j.toxcx.2024.100185. eCollection 2024 Mar.
Snakebite envenoming is a priority Neglected Tropical Disease that causes an estimated 81,000-135,000 fatalities each year. The development of a new generation of safer, affordable, and accessible antivenom therapies is urgently needed. With this goal in mind, rigorous characterisation of the specific toxins in snake venom is key to generating novel therapies for snakebite. Monoclonal antibodies directed against venom toxins are emerging as potentially strong candidates in the development of new snakebite diagnostics and treatment. Venoms comprise many different toxins of which several are responsible for their pathological effects. Due to the large variability of venoms within and between species, formulations of combinations of human antibodies are proposed as the next generation antivenoms. Here a high-throughput screening method employing antibody-based ligand fishing of venom toxins in 384 filter-well plate format has been developed to determine the antibody target/s The approach uses Protein G beads for antibody capture followed by exposure to a full venom or purified toxins to bind their respective ligand toxin(s). This is followed by a washing/centrifugation step to remove non-binding toxins and an in-well tryptic digest. Finally, peptides from each well are analysed by nanoLC-MS/MS and subsequent Mascot database searching to identify the bound toxin/s for each antibody under investigation. The approach was successfully validated to rapidly screen antibodies sourced from hybridomas, derived from venom-immunised mice expressing either regular human antibodies or heavy-chain-only human antibodies (HCAbs).
蛇咬伤中毒是一种重点被忽视的热带病，每年估计导致81,000至135,000人死亡。迫切需要开发新一代更安全、可负担且可获取的抗蛇毒血清疗法。出于这一目标，对蛇毒中特定毒素进行严格表征是开发蛇咬伤新疗法的关键。针对毒液毒素的单克隆抗体正在成为新蛇咬伤诊断和治疗开发中潜在的有力候选物。毒液包含许多不同的毒素，其中几种是其病理效应的成因。由于种内和种间毒液的巨大变异性，人源抗体组合制剂被提议作为下一代抗蛇毒血清。在此，已开发出一种高通量筛选方法，采用基于抗体的毒液毒素配体垂钓，以384孔滤板形式来确定抗体靶点。该方法使用蛋白G珠捕获抗体，随后使其接触全毒液或纯化毒素以结合各自的配体毒素。接着进行洗涤/离心步骤以去除未结合的毒素并进行孔内胰蛋白酶消化。最后，通过纳升液相色谱-串联质谱法分析每个孔中的肽段，并随后在Mascot数据库中搜索，以鉴定所研究的每种抗体结合的毒素。该方法已成功验证，可快速筛选源自杂交瘤的抗体，这些杂交瘤来自表达常规人源抗体或仅重链人源抗体（HCAbs）的经毒液免疫的小鼠。
