Detection of abrin holotoxin using novel monoclonal antibodies

Authors: He, Xiaohua; Patfield, Stephanie; Cheng, Luisa; Stanker, Larry; Rasooly, Reuven; McKeon, Thomas; Zhang, Yuzhu; Brandon, David

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2017
Publication Date: 11/28/2017
Citation: He, X., Patfield, S.A., Cheng, L.W., Stanker, L.H., Rasooly, R., McKeon, T.A., Zhang, Y., Brandon, D.L. 2017. Detection of abrin holotoxin using novel monoclonal antibodies. Toxins. 9(12):386. https://doi.org/10.3390/toxins9120386.
DOI: https://doi.org/10.3390/toxins9120386

Interpretive Summary: Abrin is a toxic protein present in rosary peas (Abrus precatorius). It shares structural and functional properties with ricin, but is four times more potent than ricin. Abrin seeds have been used in attempted suicide and unintentional ingestion by children. It has been considered as a potential bioterrorism agent due to its toxicity and easy availability. To protect U.S. food from adulteration with abrin and reduce the risk of threat to human health, it’s critical to develop a sensitive assay for detection of abrin in food. In this study, new abrin monoclonal antibodies (mAbs) and standards were developed. An immunoassay established using two of these mAbs with special characteristics was able to identify intact/active abrin in plant extracts and detects less than 1 ng/mL of abrin in non-fat and whole milk, well below the human lethal dose. These results suggest the new assay could be useful for regular food surveillance programs.

Technical Abstract: Abrin, a member of ribosome-inactivating protein family, is produced by the Abrus precatorius plant. It has been determined to have the potential to pose a severe threat to both human and animal health and is listed as a select agent by the U.S. Federal Select Agent Program. However, an immunoassay that is specific for intact abrin holotoxins and detects all 4 isoforms of abrin 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 respectively have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chain. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and five B-chain specific mAbs (Abrin-3 to Abrin-5). A sandwich ELISA, capable of detecting a mixture of 4 isoforms of intact abrin was developed using the B-chain specific mAb, Abrin-3 as a capturer and the A-chain specific mAb, Abrin-2 as a detector. The ELISA assay was shown highly sensitive and detected = 1 ng/mL of the mixture of abrin in PBS, nonfat milk, and whole milk, well below the concentrations that would pose a health concern. This ELISA was also shown to be capable of identifying native abrin present in plant extract with very low background noise. To develop standards for abrin isoforms, recombinant toxoids of four abrin isoforms (abrin-a, -b, c-, and –d) were created. The new abrin mAbs, recombinant isoforms and ELISA should be useful for developing abrin new assays and improving milk supply chain safety and security.