Botulism Diagnostics: From Clinical Symptoms toin vitroAssays

Abstract

Botulinum neurotoxin (BoNT), which cause the deadly neuroparalytic disease, botulism, is the most toxic substance known to man. BoNT can be used as potential bioterrorism agents, and therefore, pose great threat to national security and public health. Rapid and sensitive detection of BoNTs using molecular and biochemical techniques is an essential component in the diagnosis of botulism, and is yet to be achieved. The most sensitive and widely accepted assay method for BoNTs is mouse bioassay, which takes 4 days to complete. This clearly can not meet the need for clinical diagnosis of botulism, botulinum detection in field conditions, and screening of large scale samples. Consequently, the clinical diagnosis of botulism relies on the clinical symptom development, thus limiting the effectiveness of antitoxin treatment. In response to this critical need, many in vitro methods for BoNT detection are under development. This review is focused on recently developed in vitro detection methods for BoNTs, and emerging new technologies with potential for sensitive and rapid in vitro diagnostics for botulism.

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…Alternative in vitro methods for detection of BoNT have been developed to overcome some of the limitations of the mouse bioassay [23,24,25,37]. The majority of the in vitro assays developed over the last four decades are immunological methods based on the binding of an antibody to the toxin.…”

“…Finally, with the ever-increasing medical use of BoNT, its sensitive and specific detection in manufacturing processes as well as clinical research laboratories is of crucial importance. Methods of BoNT detection were recently reviewed in several excellent publications [23,24,25]. This article is intended to give the reader an update on the most recent developments and put them into the context of previously reported methods.…”

“…Despite enormous progress in the development of alternative in vitro methods for botulinum neurotoxin detection [23,24,25], the mouse lethality assay has remained the only accepted standard test to confirm active BoNTs [26]. This test is based on intraperitoneal injection of mice with dilutions of BoNT-suspected samples and subsequent observation of these mice for symptoms of botulism and, ultimately, death.…”

“…Over three decades of use for BoNT detection [45,46,47], ELISA has become by far the most commonly employed method for in vitro detection of the toxin [23,24,25]. As a means of detection, ELISA uses specific antibody binding to the toxin.…”

Improved detection of botulinum neurotoxin serotype A by Endopep–MS through peptide substrate modification

“…To overcome these limitations and reduce the use of live animals, a number of in vitro methods have been reported as alternative methods to detect BoNTs. (Cai, Singh et al 2007) The in vitro methods fall into a few major categories including immune-based assays, endoprotease activity assays and a combination immunocapture/endoprotease activity assay. Traditional enzyme-linked immunosorbent assay (ELISA) has been adapted for the detection of botulinum neurotoxins, but the method cannot ascertain whether the toxin detected is still functionally active (Ferreira, Maslanka et al 2003;Ferreira, Eliasberg et al 2004;Sharma, Ferreira et al 2006), lack the necessary sensitivity to detect toxins in many clinical samples, and have been shown to suffer from cross reactivity (Ferreira, Maslanka et al 2003).…”