Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies.

Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.

Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Physical studies on three lectins from the seeds of Abrus precatorius.

The physical properties of three lectins from the seeds of the Abrus precatorius plant, abrin C, abrin A and the Abrus agglutinin, were studied. All three exhibited similar circular dichroic (CD) spectra in the near-ultraviolet having negative maxima at 286 and 293 nm. In addition, D-galactose induced similar conformational alterations in the three proteins as observed through changes in the near-ultraviolet CD from 280 to 295 nm. The near-ultraviolet CD spectrum of the toxic subunit of abrin C was very different from that of the parent molecule. The fluorescence emission spectra of the three proteins were also studied. All exhibited fluorescence near 335 nm which is quenched 9% by galactose. Iodide quenching of fluorescence using the Stern-Volmer analysis indicated different tryptophan accessibilities in the presence and absence of D-galactose for the Abrus agglutinin. The results suggest that there is a saccharide-induced conformational change which buries several partially exposed tryptophan residues. A comparable analysis of the closely related Ricinus agglutinin revealed that its tryptophan residues are more buried than those of the Abrus agglutinin and, unlike the Abrus agglutinin, there was no saccharide-induced change in tryptophan accessibility.