Development of an In Vitro Bioassay for Clostridium botulinum Type B Neurotoxin in Foods That Is More Sensitive than the Mouse Bioassay

Abstract

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin’s light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml−1 (0.5 mouse 50% lethal dose ml−1) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.

Microarray Immunoassay Tests for Simultaneous Detection of Five Botulinum Toxins by Phosphorescence Analysis (PHOSPHAN)

“…поскольку эффективность лечения ботулизма зависит от количества токсина, попавшего в организм, и своевременности оказания медицинской помощи, то весьма актуальна разработка экспрессных, высокочувствительных и специфичных методов обнаружения. в качестве «золотого стандарта» для лабораторного выявления ботулотоксинов рассматривается метод биопробы на белых мышах, обеспечивающий порог детекции (по бта и бтв) порядка 10-30 пг/мл [6,13]. его постановка, однако, может занимать до 72 ч. другие методы (иммунохроматографический или иммуноферментный анализ, реакция пассивной гемагглютинации, молекулярные методы) более быстрые, но значительно менее чувствительные [4].…”

A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices

“…There are numerous methods (in vivo, ex vivo, and in vitro) that are currently used to detect botulinum neurotoxins and/or C. botulinum contamination. The in vivo mouse bioassay is considered the "gold standard" because of its high sensitivity (LOD ≅ 20 -30 pg) [8,9] and reliability to model all aspects of BoNT intoxication [10,11]. However, this assay is time-consuming, expensive, and requires experienced personnel and specialized facilities.…”

Monoclonal antibodies and reagents for botulinum research

“…Detection of BoNT relies on use of the 'gold standard' mouse bioassay, a highly sensitive method with detection limits of 10-20 pg/mL (Ferreira et al, 2003;Wictome et al, 1999). However, the mouse bioassay is time consuming (up to four days), requires the use of large numbers of animals, has virtually no dynamic range, and relies on death as an endpoint.…”