|SCOTCHER, MILES - Former ARS Employee|
|Cheng, Luisa Wai Wai|
|STANKER, LARRY - Former ARS Employee|
Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2019
Publication Date: 7/13/2019
Citation: Bever, C.R., Scotcher, M., Cheng, L.W., Hnasko, R.M., Stanker, L. 2019. Development and characterization of monoclonal antibodies to botulinum neurotoxin type E. Toxins. 11:407. https://doi.org/10.3390/toxins11070407.
Interpretive Summary: Botulism is a serious, often fatal neuroparalytic foodborne disease in humans and animals caused by a toxin produced by the bacterium Clostridium botulinum. Botulinum neurotoxin (BoNT) is considered the most toxic biological agent known and it is classified as a class ‘A’ bioterrorism agent. Seven different serotypes (BoNT A-G) of toxin are well documented. Serotype E outbreaks are the most prevalent type in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. The ‘gold standard’ for detection of botulinum toxin is the mouse bioassay. Simple, non-rodent-based rapid detection methods are highly desirable to monitor food and environmental samples. In this work, we describe the development and characterization of a set of monoclonal antibodies to BoNT/E, and the application of these antibodies to produce a simple, sensitive, and selective sandwich immunoassay for BoNT/E. Application of this assay to toxin spiked fish samples demonstrate the utility of this assay to measure toxin contamination in suspected food products. Our ability to rapidly screen fish products for botulinum neurotoxin serotype E improves our ability to detect this lethal foodborne agent ultimately resulting in a safer food supply.
Technical Abstract: Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A-G). Most foodborne intoxications are caused by serotypes BoNT/A and BoNT/B. BoNT/E outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the sensitive and selective detection of BoNT/E. Monoclonal antibodies (mAbs) were generated against BoNT/E by immunizing with recombinant peptide fragments of the light and heavy chains of BoNT/E. In all, 12 mAbs where characterized for binding to both the recombinant peptides and holotoxin, as well as their performance in Western blots and sandwich ELISAs. The most sensitive sandwich assay, using different mAbs for the capture and detection, exhibited a limit of detection of 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. By employing two different mAbs for capture and detection, a more standardized sandwich assay has been constructed. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals.