Vollmer Aline C, Fecher-Trost Claudia, Jung Martin, Cole Marnie, Arnst Tilman F, Flockerzi Veit, Wagmann Lea, Meyer Markus R
Experimental and Clinical Toxicology and Pharmacology, Center for Molecular Signaling (PZMS), PharmaScienceHub (PSH), Saarland University, 66421, Homburg, Germany.
Department of Medical Biochemistry and Molecular Biology, Saarland University, 66421, Homburg, Germany.
Arch Toxicol. 2025 Jul 11. doi: 10.1007/s00204-025-04132-x.
The analysis of highly toxic proteins such as abrin and ricin is challenging but comprehensive analytical methods are essential for their unambiguous identification after ingestion. This study pursued three primary aims at detecting abrin and ricin in human biosamples while ensuring that laboratory staff remain protected from direct exposure to these toxic proteins. First, two polyclonal antibodies (pAB) against specific peptides of abrin-A and ricin should be produced. Thereby, antibody epitope mapping was performed, which proved that both pAB recognize specifically their target peptide. Second, an affinity column chromatography-based assay was developed, and finally, the generated pAB should be tested using two different approaches (A and B) for their application in mass spectrometry (MS)-based bioanalytical workflows. Approach A used blood and urine samples submitted to the author's laboratory after suspected ricin intake. Samples were prepared for nanoLC-MS analysis using affinity column chromatography, gel electrophoresis, and overnight trypsin digestion. Analysis resulted in the confirmation of ricin presence in both plasma and urine. Approach B included the enrichment of an abrin-A-peptide and ricin-peptide using affinity column chromatography directly followed by LC-Orbitrap MS with a detection limit of at least 5 ng/mL in plasma and validation according to international recommendations. Since approach B is much more time-efficient and can be applied throughout laboratories due to the less equipment required, this strategy deserves further focus, especially in a clinical setting.
对相思子毒素和蓖麻毒素等高毒性蛋白质进行分析具有挑战性，但全面的分析方法对于摄入后明确鉴定它们至关重要。本研究有三个主要目标，即在检测人体生物样本中的相思子毒素和蓖麻毒素的同时，确保实验室工作人员免受这些有毒蛋白质的直接接触。首先，应制备两种针对相思子毒素-A和蓖麻毒素特定肽段的多克隆抗体（pAB）。由此进行了抗体表位作图，结果证明两种pAB均能特异性识别其靶肽段。其次，开发了一种基于亲和柱色谱的检测方法，最后，应使用两种不同方法（方法A和方法B）测试所产生的pAB在基于质谱（MS）的生物分析工作流程中的应用。方法A使用在怀疑摄入蓖麻毒素后提交给作者实验室的血液和尿液样本。使用亲和柱色谱、凝胶电泳和过夜胰蛋白酶消化对样本进行纳米液相色谱-质谱分析制备。分析结果证实血浆和尿液中均存在蓖麻毒素。方法B包括使用亲和柱色谱直接富集相思子毒素-A肽段和蓖麻毒素肽段，随后进行液相色谱-轨道阱质谱分析，血浆中的检测限至少为5 ng/mL，并根据国际建议进行验证。由于方法B效率更高，且所需设备较少，可在各实验室应用，因此该策略值得进一步关注，尤其是在临床环境中。
