Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Summary

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices. Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Abstract

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

Introduction

Botulinum neurotoxins (BoNTs) are the deadliest substances known, with intravenous human lethal doses estimated at 1-3 ng/kg^1,2. Seven structurally similar serotypes of BoNT, labeled A through G, exist, each consisting of a heavy chain domain responsible for cell binding, uptake, and translocation into the cytosol and a light chain that encodes a zinc endopeptidase^3-5. The exquisite toxicity of BoNT results from, in part, its specific binding and entry into motor neurons at the neuromuscular junction⁶. Once inside the neuron, the light chain endopeptidase specifically cleaves one or more of the soluble N-ethylmaleimide-sensiti....

Protocol

1. Preparation of Assay Reagents

1. Thaw 200x dithiothreitol (DTT), 10x Matrix Binding buffer (10x Binding buffer hereafter), 10x Neutralization buffer (food or pH imbalanced samples only), and 10x BoTest Reaction buffer (10x Reaction buffer hereafter) at room temperature (RT) for 15 min or until completely thawed. See Table 1 for a list of buffers and reagents used in this protocol. See Table 2 for a list of materials and equipment required for this protocol.
2. Vortex the thawed buffers for 5 sec to mix. 10x Neutralization buffer, 200xDTT, and 10x Reaction buffer should appear clear while the 10x Binding buffer will ....

Results

A diagram summarizing the steps in the described protocol is shown in Figure 2. The assay requires between 4-26 hr to complete depending on sample type and desired assay sensitivity, but only ~2 hr of hands-on time. The assay is performed in 96-well plates and, depending on the type of testing being performed, allows triplicate testing of up to 20 samples including standards per plate.

Figure 3 shows representative assay results using BoNT/A holotoxin spiked i.......

Discussion

This protocol describes procedures for quantifying BoNT/A complex, holotoxin, or Clostridium culture supernatant in complex matrices. The protocol is the same, however, when testing other BoNT serotypes (e.g. BoNT/B, E, and F) with their respective Matrix assays^56,60, although assay sensitivity will vary across serotypes and assays. This protocol does not account for every type of sample possible and some modifications may be required depending on the specific sample composition and desired a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BoTest Matrix A Botulinum Neurotoxin Detection Kit	BioSentinel	A1015	Detection kits for BoNT/B and F are also available.
Varioskan Flash fluorescence microplate reader	Thermo Fisher Scientific	5250040	Most monochromator- or filter-based units with 434 nm excitation and 470 nm and 526 nm emission capability can be used.
96-well Magnetic Bead Separation Plate	V&P Scientific	VP771H	Other magnetic plates may be used, but the plate should be designed to separate the beads to the side of the well.
Magnetic Bead-Compatible Plate Washer	BioTek	ELx405 VSRM	Optional, only required for automated plate washing.  Other magnetic bead-compatible plate washers may also be used, but should be tested before use.
Microcentrifuge			Optional, only required for samples needing centrifugation.
MixMate plate mixer	Eppendorf	22674200
Orbital Shaker			Used at room temperature or at 25 °C If temperature control is available
EDTA-free Protease Inhibitor Tablets	Roche	4693132001	Only required for food or environmental testing. Protease inhibitors must be EDTA-free.
BoNT/A	Metabiologics		Optional, only required for standardization and quantification purposes
Black, Flat-bottomed 96-well Plates	NUNC	237105	Plates should not be treated
96-well Plate Sealing Tape	Thermo Fisher Scientific	15036