High throughput identification of human monoclonal antibodies and heavy-chain-only antibodies to treat snakebite

IF 3.6 Q2 TOXICOLOGY
Julien Slagboom , Abigail H. Lewis , Wietse M. Schouten , Rien van Haperen , Mieke Veltman , Mátyás A. Bittenbinder , Freek J. Vonk , Nicholas R. Casewell , Frank Grosveld , Dubravka Drabek , Jeroen Kool
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Abstract

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).

Abstract Image

高通量鉴定治疗蛇咬伤的人类单克隆抗体和纯重链抗体
蛇咬伤是一种优先防治的被忽视热带病,估计每年造成 81,000 至 135,000 人死亡。开发新一代更安全、更经济、更易获得的抗蛇毒血清疗法迫在眉睫。为实现这一目标,对蛇毒中的特异性毒素进行严格鉴定是开发新型蛇咬伤疗法的关键。针对毒液毒素的单克隆抗体正在成为开发新型蛇咬伤诊断和治疗方法的有力候选药物。毒液由多种不同的毒素组成,其中有几种是造成病理效应的原因。由于毒液在物种内部和物种之间存在很大的变异性,因此有人建议将人类抗体的组合配方作为下一代抗蛇毒血清。在此,我们开发了一种高通量筛选方法,采用基于抗体的毒液毒素配体钓法,在 384 滤孔板格式中确定抗体靶标。该方法使用蛋白 G 珠捕获抗体,然后将其暴露于全毒液或纯化毒素中,以结合各自的配体毒素。然后进行洗涤/离心步骤以去除不结合的毒素,并进行孔内胰蛋白酶消化。最后,通过纳米液相色谱-质谱-质谱(nanoLC-MS/MS)和随后的 Mascot 数据库搜索分析各孔中的肽段,以确定所研究抗体的结合毒素。该方法已成功用于快速筛选来自杂交瘤的抗体,杂交瘤来自表达普通人类抗体或纯重链人类抗体(HCAbs)的毒液免疫小鼠。
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来源期刊
Toxicon: X
Toxicon: X Pharmacology, Toxicology and Pharmaceutics-Toxicology
CiteScore
6.50
自引率
0.00%
发文量
33
审稿时长
14 weeks
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