A Label Free Colorimetric Assay for the Detection of Active Botulinum Neurotoxin Type A by SNAP-25 Conjugated Colloidal Gold

Jennifer Halliwell,Christopher Gwenin

doi:10.3390/toxins5081381

Abstract

Botulinum neurotoxins are one of the most potent toxins known to man. Current methods of detection involve the quantification of the toxin but do not take into account the percentage of the toxin that is active. At present the assay used for monitoring the activity of the toxin is the mouse bioassay, which is lengthy and has ethical issues due to the use of live animals. This report demonstrates a novel assay that utilises the endopeptidase activity of the toxin to detect Botulinum neurotoxin in a pharmaceutical sample. The cleaving of SNAP-25 is monitored via UV-Visible spectroscopy with a limit of detection of 373 fg/mL and has been further developed into a high throughput method using a microplate reader detecting down to 600 fg/mL of active toxin. The results show clear differences between the toxin product and the placebo, which contains the pharmaceutical excipients human serum albumin and lactose, showing that the assay detects the active form of the toxin.

Highlights

The method currently used for determining the concentration of toxin in a sample is the mouse bioassay
An assay which outperforms current limits of detection and takes minutes to perform would fit a gap in the market having uses in the pharmaceutical industry for batch quality control, as a point of care clinical sensor in suspect botulism cases as well as in biosecurity applications [6]
UV-Visible spectrum of coated and uncoated gold particles were measured before and after salt was added to prove that the SNAP-25 bound with the gold surface (Figure 1)

Summary

Introduction

The method currently used for determining the concentration of toxin in a sample is the mouse bioassay. This assay involves injecting the mice with a sample of the toxin and observing them for Toxins 2013, 5 symptoms of the disease over a period of up to four days [1]. There is demand for alternative methods to reduce and replace the use of live animals and many botulinum assays are currently being developed including ELISA and fluorescent endopeptidase assays which outperform the mouse bioassay and takes 1–3 h [3,4,5]. An assay which outperforms current limits of detection and takes minutes to perform would fit a gap in the market having uses in the pharmaceutical industry for batch quality control, as a point of care clinical sensor in suspect botulism cases as well as in biosecurity applications [6].

Methods

Results

Conclusion