Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies

Abstract

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.

Novel Phage Display-Derived Anti-Abrin Antibodies Confer Post-Exposure Protection against Abrin Intoxication

“…Over the last decade, several anti-abrin monoclonal antibodies (mABs) were isolated, several targeting ATA and only one targeting ATB. The mABs were found to be specific and were successfully implemented in several formats of toxin detection [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. The effectiveness of anti-abrin mABs neutralization in vivo was demonstrated only in two cases, where the antibodies were given as a prophylactic treatment [ 6 , 8 ], thus highlighting the need for potent anti-abrin antibodies that will provide effective post-exposure therapy.…”

Differentiation, Quantification and Identification of Abrin and Abrus precatorius Agglutinin

“…Regarding assay sensitivity, the two stationary ELISAs developed in this work delivered excellent detection limits: for the abrin-specific ELISA, an LOD of 22 pg/mL, and for the APA-specific ELISA, an LOD of ~35 pg/mL were determined. In comparison, previously reported ELISAs for abrin delivered detection limits between 100 pg/mL and 7800 pg/mL [ 31 , 32 , 33 , 34 , 57 ]; therefore, the current work significantly advances the field, offering tools with higher sensitivity as well as increased specificity and selectivity.…”

“…It is worthy of note that by combining a capture mAb directed against the B-chain of abrin (AP430) with a detection mAb recognizing the A-chain (AP3202), the abrin-specific ELISA detected only the intact A-B toxin. In previous works, the issue of mAb / ELISA selectivity for abrin versus APA has rarely been addressed; only a few groups have reported either cross-reactive or specific mAbs for abrin and APA derived from mouse or llama [ 32 , 56 , 57 ]. In a broader context, sandwich ELISAs able to differentiate between related toxin subtypes or isolectins have been successfully established based on highly specific mAbs directed against unique domains found in the related molecules (e.g., for ricin/RCA120 or for the related botulinum neurotoxins BoNT/C, CD, DC, and D [ 47 , 48 ]).…”

“…Only a few immunological and MS-based assays have been described that directly detect the presence of the toxic compound abrin. Immunological assays such as sandwich enzyme-linked immunosorbent assays (ELISAs) can detect between 100 to 4000 pg/mL abrin and often work quite well with complex matrices such as beverages or foods [ 31 , 32 , 33 , 34 ]. Methods for on-site detection such as lateral flow assays (LFAs) can reach detection limits between 100 and 50,000 pg/mL but deliver results in less than an hour compared to the approximately four to six hours required for a conventional sandwich ELISA performed in microtiter plates [ 34 , 35 , 36 , 37 ].…”

Biological Toxins as the Potential Tools for Bioterrorism

“…In the cells’ cytosol, chain A cleaves the C-N bond in adenines, located in the small subunit of the ribosome 28S rRNA at the position A4324. Adenine depurination destabilizes the rRNA preventing the binding of ribosomes to elongation factors, and this results in inhibition of protein synthesis [ 90 ].…”