Aim: To develop a novel assay technique for the botulinum neurotoxin family (BoNTs) which is dependent on both the endopeptidase and receptor-binding activities of the BoNTs and which is insensitive to antigenic variation with the toxin family. Methods and Results: An endopeptidase activity, receptor-binding assay (EARB assay) has been developed which captures biologically active toxin from media using brain synaptosomes. After capture, the bound toxin can be incubated with its substrate, and cleavage detected using serotype-specific antibodies raised against the cleaved product of each toxin serotype. The EARB assay was assessed using a range of BoNT serotypes and subtypes. For BoNT/A, detection limits for subtypes A1, A2 and A3 were 0·5, 3 and 10 MLD50 ml-1, respectively. The limit of detection for BoNT/B1 was 5 MLD50 ml-1 and a novel antibody-based endopeptidase assay for BoNT/F detected toxin at 0·5 MLD50 ml-1. All these BoNTs can be captured from media containing up to 10% serum without loss of sensitivity. BoNT/A 1 could also be detected in dilutions of a lactose- containing formulation similar to that used for clinical preparations of the toxin. Different serotypes were found to possess different optimal cleavage pHs (pH 6·5 for A1, pH 7·4 for B1). Conclusions: The EARB assay has been shown to be able to detect a broad range of BoNT serotypes and subtypes from various media. Significance and Impact of the Study: The EARB assay system described is the first convenient in vitro assay system described which is requires multiple functional biological activities with the BoNTs. The assay will have applications in instances where it is essential or desirable to distinguish biologically active from inactive neurotoxin.
Bibliographical noteFunding Information:
We would like to thank the Research Incentive Fund of the Clinical Hospital of Porto Alegre (FIPE/HCPA - project number 12-0116) for the financial support, and the Coordination for the Improvement of Higher-Level Personnel (CAPES), the Research Support Foundation of Rio Grande do Sul (FAPERGS), the National Council of Research Development (CNPq), the Federal University of Rio Grande do Sul (UFRGS), the Experimental Hepatology and Gastroenterology Laboratory (HCPA/UFRGS) and the Lutheran University of Brazil (ULBRA).
- Mechanism of action
- Rapid techniques