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.

FIG. 1

Assay format for detection of C. botulinum BoNT/B. , immobilized monoclonal antibody; , neurotoxin; , biotinylated substrate; ▄, streptavidin.

FIG. 2

Assay for detection of C. botulinum BoNT/B. Data were obtained from 33 assays in which we used HZ buffer containing 5% FCS spiked with purified BoNT/B. The error bars indicate standard deviations (n − 1). The dotted line shows the detection limit at 0.5 absorbance unit above the background value. OD 450nm, optical density at 450 nm.