Polyclonal antibodies towards abrin and ricin-design and potential application for mass spectrometry-based analysis of human biosamples

Abstract

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 pro teins. 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.