Advancement of abrin toxin bioforensics capabilities

Abstract

Abrin toxin, located in the seeds of Abrus precatorius, is a potent Type II Ribosome-Inactivating Protein (RIP) A-B subunit toxin that has acquired a heightened biothreat profile over the past decade. Significant research efforts must be made in order to develop abrin bioforensics diagnostic response capabilities to the level which is currently in place for ricin, another Type II RIP toxin with similar molecular structure and mechanism of toxicity. To serve this endeavor, three interrelated investigations have been carried out. The first study addresses the need to expand the supply of anti-abrin monoclonal antibody (mAb) reagents. Monoclonal antibodies were developed from hybridomas immunized with synthetic peptides derived from in silico prediction of abrin B-cell epitopes. Two epitopes produced nine abrin-reactive mAbs. Secondly, an attenuated, antigenically-faithful recombinant proabrin holotoxoid was expressed and purified using the baculovirus system for purposes as both an immunogen in future hybridoma experiments, as well as a non-toxic ‘safe antigen’ diagnostic reagent standard. The reduction in toxicity of proabrin relative to wild-type abrin was 108-fold in cell-free translation assay, and 291- to 302-fold in cytotoxicity assays. Finally, the third study combined shotgun proteomics with multivariate analysis differentiation methods in order to retrospectively identify six toxin extraction protocols with further sub-variation of reagent source. Differentiation was based solely on forensic proteomic signatures of carryover seed proteins. A 5-way hierarchical sPLS-DA model correctly classified samples into 8 extraction categories with 100 percent accuracy. Continued assessment of FASP LC-MS/MS and sPLS-DA modelling for attribution of plant toxin extraction methods may lead to establishment of a novel bioforensics diagnostic platform. Together, these three studies serve to advance Canadian bioforensics capabilities and to provide a foundation for future work.