The authors would like to thank H. Olivares and D. Ruge for valuable discussions and advice. This research was supported in part by a NSF SBIR award (IIP-1127245 to BioSentinel Inc.) and a Department of Defense contract (W81XWH-07-2-0045 to BioSentinel Inc.).

1. Preparation of Assay Reagents
<ol>
	<li>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.</li>
	<li>Vortex t...

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 assays56,60, 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...

Botulinum neurotoxins (BoNTs) are the deadliest substances known, with intravenous human lethal doses estimated at 1-3 ng/kg1,2. 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 endopeptidase3-5. 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-sensitive factor attachment protein receptor (SNARE) proteins required for vesicle fusion, inhibiting neurotransmitter release and leading to flaccid paralysis7-14. Commonly known as the disease &#34;botulism,&#34; paralysis of the diaphragm and intercostal muscles by BoNT ultimately results in respiratory failure and death unless early diagnosis and treatment are received.
Human foodborne botulism is most commonly associated with BoNT serotypes A, B, E, and F (BoNT/A, BoNT/B, etc.) and usually results from the ingestion of contaminated food15,16; although, several cases of wound botulism were reported amongst intravenous drug users17,18. In the United States, infant botulism resulting from the ingestion of Clostridium spores by children under the age of one is the most common form of botulism19-21. However, foodborne BoNT outbreaks resulting from improper home canning and food processing were reported in both the United States and abroad. Between 2000-2009, at least 338 cases of foodborne botulism were reported worldwide including six fatalities22. The ability to rapidly and sensitively detect foodborne botulism outbreaks is a critical indication that could aid early diagnosis23,24. Furthermore, detection methods that allow cost-effective and routine food testing will lead to improved food security.
BoNT&#39;s neuronal specificity and long biological half-life also makes it a potent therapeutic. In the United States, BoNT-based drugs are approved by the Food and Drug Administration for the treatment of cosmetic conditions and neuromuscular-related disorders including glabellar lines, cervical dystonia, migraine headaches, overactive bladder, and strabismus. Numerous &#34;off-label&#34; applications are documented, including high-dose treatments for severe muscle dysfunction25-28. Accurate toxin quantification is critical for correct dosing, as underdosing may lead to ineffective treatment while overdosing puts patients at risk of potentially harmful side effects. Unfortunately, no standardized potency assay protocol is shared across manufacturers, resulting in unit definition inequalities between BoNT-based drug products29-31.
The standard test for BoNT is the mouse bioassay in which BoNT-containing samples are injected intraperitoneally into mice and the numbers of deaths recorded over 1-7 days16,32,33. The mouse bioassay is very sensitive with limits of detection (LOD) of 5-10 pg BoNT/A34; however, ethical concerns over animal use, the high cost of training personnel and maintaining animal facilities, long assay times, and the lack of standardized protocols resulted in calls to develop standardized, animal-free BoNT testing and quantification methods35-39. Recently, several alternate BoNT quantification methods were developed that offer mouse or near-mouse bioassay sensitivity40-49. These methods commonly use fluorescence, mass-spectrometry, or immunological methods and offer assay times considerably shorter than the mouse bioassay without animal use. Mass-spectrometry approaches combined with immunological techniques were shown to detect and quantify BoNT contained in food and other complex samples; however, personnel training requirements and specialized equipment limit these assays50-55. Most other alternate assays are not readily applicable to complex sample testing or lack the throughput required for routine BoNT testing. The highly variable nature of food sample viscosity, pH, salt content, and matrix constituents presents an especially difficult challenge when trying to develop in vitro assay methods with sensitivity to match the extreme potency of BoNT. Furthermore, even simple and relatively benign buffer systems, such as those resulting from resuspension of BoNT-based drug products, contain salt, albumin, and sugar stabilizers (i.e. excipients) that significantly impact in vitro BoNT potency56. Toxin purification is required for accurate activity testing of all but the simplest of samples56-59.
The BoTest Matrix assays were designed for rapid, high-throughput, and consistent quantification of BoNT from highly complex samples using equipment commonly found in research laboratories56,60. These assays use paramagnetic beads covalently linked to serotype-specific anti-BoNT antibodies to bind and sequester BoNT out of a sample and then remove interfering matrix compounds by washing. Following washing, bound BoNT proteolytic activity is then quantified in an optimized reaction buffer using a reporter compatible with the BoNT serotype being tested. These reporters are fluorogenic proteins consisting of a N-terminal cyan fluorescent protein (CFP) moiety and a C-terminal yellow fluorescent protein derivative (Venus) moiety linked by a BoNT substrate, SNAP25 residues 141-206 or synaptobrevin residues 33-94 constituting the BoTest A/E or B/D/F/G reporters, respectively45. Reporter cleavage by BoNT is monitored using F&#246;rster resonance energy transfer (FRET). When the reporter is intact, excitation of CFP results in FRET to Venus, quenching CFP emission and exciting Venus emission. Cleavage of the reporter by BoNT prevents FRET, leading to an increase in CFP emission and decrease in Venus emission. BoNT activity can then be quantitatively measured using the ratio of the CFP and Venus emissions. LOD below 3 pg are possible from a wide range of foods using a high-throughput 96-well plate format56. Increased sensitivity can be obtained using larger sample volumes since the assay allows concentration of the toxin on the bead surface.
The BoTest Matrix assays for BoNTs A, B, E, and F were developed and tested with food, pharmaceutical, and environmental samples56,60. Here, we describe procedures for executing these assays for the detection of BoNT in low complexity (e.g. pharmaceutical, BoNT in buffer) and high complexity (e.g. food, environmental) samples. Specific processing methods for several sample types are addressed in this protocol and sample types not described here can usually be adapted using a combination of the presented methods. The protocol was developed and tested with BoNT/A but is adaptable to other BoNT serotypes using their respective assays as demonstrated elsewhere56,60.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">BoTest Matrix A Botulinum Neurotoxin Detection Kit</td>
			<td style="width:107px;">BioSentinel</td>
			<td style="width:104px;">A1015</td>
			<td style="width:245px;">Detection kits for BoNT/B and F are also available.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">Varioskan Flash fluorescence microplate reader</td>
			<td style="width:107px;">Thermo-Fisher Scientific</td>
			<td style="width:104px;">5250040</td>
			<td style="width:245px;">Most monochromator- or filter-based units with 434 nm excitation and 470 and 526 nm emission capability can be used.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">96-well Magnetic Bead Separation Plate</td>
			<td style="width:107px;">V&#38;P Scientific&#160;</td>
			<td style="width:104px;">VP771H</td>
			<td style="width:245px;">Other magnetic plates may be used, but the plate should be designed to separate the beads to the side of the well.</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:192px;">Magnetic Bead-Compatible Plate Washer</td>
			<td style="width:107px;">BioTek&#160;</td>
			<td style="width:104px;">ELx405 VSRM</td>
			<td style="width:245px;">Optional, only required for automated plate washing.&#160; Other magnetic bead-compatible plate washers may also be used, but should be tested before use.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Microcentrifuge</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for samples needing centrifugation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:192px;">MixMate plate mixer</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:104px;">22674200</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">Orbital Shaker</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Used at room temperature or at 25 &#176;C If temperature control is available</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">EDTA-free Protease Inhibitor Tablets</td>
			<td style="width:107px;">Roche</td>
			<td style="width:104px;">4693132001</td>
			<td style="width:245px;">Only required for food or environmental testing.&#160; Protease inhibitors must be EDTA-free.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">BoNT/A&#160;</td>
			<td style="width:107px;">Metabiologics</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for standardization and quantification purposes&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Black, Flat-bottomed 96-well Plates</td>
			<td style="width:107px;">NUNC</td>
			<td style="width:104px;">237105</td>
			<td style="width:245px;">Plates should not be treated</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">96-well Plate Sealing Tape</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:104px;">15036</td>
			<td style="width:245px;"></td>
		</tr>
	</tbody>
</table>

isolation and quantification of botulinum neurotoxin from complex matrices using the botest matrix assays

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&#39;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&#39;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.

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...

Watch this Scientific Journal Video about Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays at JoVE.com

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

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;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.

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

isolation-quantification-botulinum-neurotoxin-from-complex-matrices

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JoVE Journal

Neuroscience

15.8K Views.  BioSentinel Inc., Madison, WI. 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.