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-sensitive factor attachment protein receptor (SNARE) proteins required for vesicle fusion, inhibiting neurotransmitter release and leading to flaccid paralysis^7-14. Commonly known as the disease "botulism," 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 food^15,16; although, several cases of wound botulism were reported amongst intravenous drug users^17,18. 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 botulism^19-21. 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 diagnosis^23,24. Furthermore, detection methods that allow cost-effective and routine food testing will lead to improved food security.

BoNT'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 "off-label" applications are documented, including high-dose treatments for severe muscle dysfunction^25-28. 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 products^29-31.

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 days^16,32,33. 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 methods^35-39. Recently, several alternate BoNT quantification methods were developed that offer mouse or near-mouse bioassay sensitivity^40-49. 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 assays^50-55. 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 samples^56-59.

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 laboratories^56,60. 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 Förster 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 samples^56,60. 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 elsewhere^56,60.

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 have a cloudy appearance. Warm the 10x Reaction buffer for 5 min at 37 °C and repeat vortexing if it appears cloudy following thawing.
3. Generate 3.8 ml of 1x Reaction buffer.
   1. Label a 15 ml conical tube "1x Reaction buffer" and add 3.42 ml molecular biology grade water, 380 μl 10x Reaction buffer, and 19 μl 200x DTT. Mix buffer well by inversion.
   2. Cut a single EDTA-free protease inhibitor tablet in quarters using a clean razor blade and add one quarter of the tablet to the 1x Reaction buffer (the remaining tablet portion can be stored at 4 °C for later use). N.B. Protease inhibitors may not be necessary if using purified toxin and simple buffers (e.g. pharmaceutical samples).
   3. Vortex the 1x Reaction buffer until the protease tablet is fully dissolved.
4. Warm the Matrix A beads (IP-A beads hereafter) for 20 min at RT.
5. Thaw the BoTest A/E reporter (A/E reporter hereafter) at RT protected from light.
6. Label a 15 ml conical tube "0.5 μM A/E reporter" and add 45 μl of the 20 μM A/E reporter stock to 1.8 ml 1x Reaction buffer. Mix thoroughly by pipetting and store on ice protected from light.

2. Standard Curve Sample Generation

The standard curve described here spans 10-30,000 mLD₅₀/g food or per ml buffer in half-log dilutions (Table 3). The end-user is free to use alternate concentrations as applicable.

1. Spiking and incubating the matrices with reference material. Generate the standard curve using a diluent of the same matrix as the unknown, if possible, to reduce matrix effects. Use gelatin phosphate buffer (GPB) or phosphate-buffered saline (PBS) as a diluent if additional, BoNT-negative matrix is not available (e.g. field or BoNT outbreak sample testing). Generate the standard curve by spiking BoNT/A into the matrix of choice following the appropriate protocol below.
   1. Low complexity samples (e.g. PBS, GPB, or pharmaceutical)
      1. Add 10 ml of the appropriate buffer to a 15 ml conical tube and set aside. This sample will be used as diluent for the standard curve and unknown dilutions (Section 4).
      2. Add 1.2 ml buffer to a microcentrifuge tube.
      3. CAUTION: This step uses BoNT and extreme caution must be used when handling and disposing of any reagents and materials that come in contact with the toxin. Use of personal protective equipment and proper disposal of all materials must be performed according the US Department of Labor OSHA guidelines for BoNT (http://www.osha.gov/SLTC/botulism/index.html). Add BoNT/A to the 1.2 ml sample such that the final concentration is 30,000 mLD₅₀/ml (36,000 mLD₅₀ total).
   2. Liquid food samples (or other complex liquid samples) Note: Initial testing is recommended to determine the volume of supernatant recovered from clarified samples as the particulate matter (e.g. pulp) in liquid matrices will vary. This protocol assumes at least 1.2 ml of clarified supernatant will be recovered from a 1.4 ml sample. Increase sample size if necessary.
      1. Add 10 ml liquid food to a 15 ml conical tube and set it aside. This sample will be used as diluent for the standard curve and unknown dilutions (Section 4).
      2. Weigh an empty 1.5 ml microcentrifuge tube and record its mass.
      3. Add 1.4 ml of the liquid food to the weighed microcentrifuge tube.
      4. Reweigh the microcentrifuge tube and calculate the mass of the added food sample by subtracting the mass of the empty tube.
      5. CAUTION: This step uses BoNT and extreme caution must be used when handling and disposing of any reagents and materials that come in contact with the toxin. Use of personal protective equipment and proper disposal of all materials must be performed according the US Department of Labor OSHA guidelines for BoNT (http://www.osha.gov/SLTC/botulism/index.html). Add BoNT/A to the 1.4 ml sample at a final concentration of 30,000 mLD₅₀/g food.
      6. Incubate the diluent and spiked samples at RT or 4 °C for 2 hr to give the BoNT time to interact with the food matrix, mimicking a natural contamination.
   3. Solid food samples (or other solid samples) Note: Initial testing is recommended to determine the volume of supernatant recovered from homogenized and clarified samples following the addition of 1 ml GPB/g food. This protocol assumes that at least 1.2 ml clarified supernatant will be recovered from a 2 g sample. Increase sample size if necessary.
      1. Weigh out 10 g solid sample into a 50 ml conical tube and set aside. This will be used as diluent.
      2. Weigh out 2 g solid food sample into a second 50 ml conical tube.
      3. CAUTION: This step uses BoNT and extreme caution must be used when handling and disposing of any reagents and materials that come in contact with the toxin. Use of personal protective equipment and proper disposal of all materials must be performed according the US Department of Labor OSHA guidelines for BoNT (http://www.osha.gov/SLTC/botulism/index.html). Add BoNT/A to the surface of the 2 g sample to a final concentration of 30,000 mLD₅₀/g food (60,000 total mLD₅₀).
      4. Incubate the diluent and spiked samples at RT or 4 °C for 2 hr to give the BoNT time to interact with the food matrix, mimicking a natural contamination.
2. Sample homogenization and buffer adjustment. Process the spiked standard curve sample and diluent generated above according to sample type.
   1. Low complexity samples
      1. No further sample processing is necessary.
   2. Liquid food samples (or other complex liquid samples)
      1. No sample homogenization is necessary.
      2. Add 140 μl 10x Neutralization buffer to the 1.4 ml spiked sample and 1 ml 10x Neutralization buffer to the 10 ml diluent sample. Mix samples well by inversion.
      3. Partially clarify both samples by centrifuging for 10 min at 6,000 x g and 4 °C. Immediately remove the supernatants and transfer to new tubes.
   3. Solid food samples (or other solid samples)
      1. Add 2 ml GPB (1 ml GPB/g food) to the 2 g BoNT/A-spiked solid food sample and 10 ml GPB to the 10 g diluent sample.
      2. Homogenize the samples using a pestle until thoroughly blended. Depending on the nature of the sample, there may be small chunks of material that cannot be homogenized, which is acceptable. Alternately, mechanical homogenization methods may be used. Use of a blender is not recommended as it may inactivate as well as aerosolize the toxin.
      3. Add 1/10th volume of 10x Neutralization buffer to the diluent sample based on the approximate total volume (e.g. 2 ml if the volume is 20 ml). Extrapolate the total volume of the BoNT/A-spiked sample and add 1/10th volume of 10x Neutralization buffer. Mix samples well by inversion.
      4. Partially clarify both samples by centrifuging for 10 min at 6,000 x g and 4 °C. Immediately remove the supernatants and transfer to new tubes.
3. Prepare standard curve serial dilutions for testing
   1. Using the BoNT/A-spiked standard curve sample as D1 and the nonspiked sample as diluent, generate the remaining standard curve samples in 1.5 ml microcentrifuge tubes according to Table 3.

3. Prepare Unknown Samples

This section can be completed in parallel to section 2.

1. Determine the number and dilutions of unknowns. Test unknowns in triplicate if possible.
   1. For qualitative assays, run the unknowns without any further dilution than required to process the sample as described below.
   2. For quantitative assays, prepare at least two 1:10 dilution samples, as described below, to ensure that one or more samples fall within the linear range of the assay response. Generate dilutions using a diluent of the same matrix as the unknown, if possible, to reduce matrix effects as described in Section 3. Otherwise, use PBS or GPB as the diluent.
2. Generating and diluting unknown samples according to sample type.
   1. Low complexity unknowns (e.g. PBS, GPB, or pharmaceutical)
      1. Add at least 750 μl unknown to a microcentrifuge tube to generate Unknown dilution 1.
      2. Add 675 μl diluent to two microcentrifuge tubes labeled Unknown dilution 2 and 3. The diluent will be the same material used for standard curve generation.
      3. Serially dilute dilution 1 by transferring 75 μl of dilution 1 into the dilution 2 tube and mixing.
      4. Serially dilute dilution 2 by transferring 75 μl of dilution 2 into the dilution 3 tube and mixing.
   2. Liquid food unknowns (or other complex liquid samples) Note: Initial testing is recommended to determine the volume of supernatant recovered from clarified samples as discussed in Section 3. Increase sample size if necessary.
      1. Add ≥ 875 μl of the liquid unknown to a microcentrifuge tube.
      2. Add 1/10th volume of 10x Neutralization buffer to the sample (e.g. 87.5 μl for a 875 μl sample).
      3. Partially clarify the sample by centrifuging for 10 min at 6,000 x g and 4 °C. Immediately transfer the supernatant to a new tube. This is Unknown dilution 1.
      4. Add 675 μl diluent to two tubes labeled Unknown dilution 2 and 3. The diluent will be the same processed material used for standard curve generation.
      5. Serially dilute dilution 1 by transferring 75 μl of dilution 1 into the dilution 2 tube and mixing.
      6. Serially dilute dilution 2 by transferring 75 μl of dilution 2 into the dilution 3 tube and mixing.
   3. Solid food unknowns (or other solid samples) Note: Initial testing is recommended to determine the volume of supernatant recovered from clarified samples as discussed in Section 3. Increase sample size if necessary.
      1. Weigh out 2 g solid unknown sample into a 50 ml conical tube.
      2. Add 2 ml GPB (1 ml GPB/g food) to the 2 g solid sample.
      3. Homogenize the samples as described in  Section 3.2.3.2.
      4. Add 1/10th volume of 10x Neutralization buffer to the sample based on the approximate total volume (e.g. 0.4 ml if the volume is 4 ml). Mix samples well by inversion.
      5. Partially clarify the sample by centrifuging for 10 min at 6,000 x g and 4 °C. Immediately transfer ≥750 μl supernatant to a microcentrifuge tube. This is Unknown dilution 1.
      6. Add 675 ml diluent to two tubes labeled Unknown dilution 2 and 3. The diluent will be the same processed material used for standard curve generation.
      7. Serially dilute dilution 1 by transferring 75 μl of dilution 1 into the dilution 2 tube and mixing.
      8. Serially dilute dilution 2 by transferring 75 μl of dilution 2 into the dilution 3 tube and mixing.

4. Final Sample Clarification

If testing liquid or solid food samples, centrifuge all samples for 5 min at ≥14,000 x g in a microcentrifuge to fully clarify the samples. Immediately remove the supernatants and transfer to new tubes.

5. Plate Setup and BoNT/A Pull Down

1. The specific plate layout is application dependent; however, do not use the outside wells so as to avoid edge effects. Each unknown sample and standard curve sample D1-D8 requires 3 wells while sample D9 requires 6 wells. A suggested plate layout is shown in Figure 1.
2. Add 20 μl 10x Binding buffer to each well to be used.
3. Add 200 μl of each clarified dilution and unknown to each of three wells (six wells for D9) for triplicate testing. Mix the plate for 10 sec on a microplate mixer.
4. Add the IP-A beads.
   1. Vortex the IP-A beads for 10 sec at the highest speed. Continue vortexing if beads are not fully resuspended and homogeneous.
   2. Pipette 20 μl IP-A beads to each sample well.
   3. Mix the plate for 30 sec on a microplate mixer.
5. Incubate the plate using a rotating plate incubator for 2 hr at 750 rpm, 25 °C or RT. Assay performance is highly dependent on generating and maintaining the IP-A bead suspension during all incubation steps. Always resuspend beads with a microplate mixer after pelleting and maintain the suspension using an orbital microtiter plate shaker during all incubation steps.

6. Plate Washing and Bead Resuspension

1. Wash the plates either by hand or by using a magnetic bead-compatible automated plate washer. An automated plate washer configured for magnetic beads greatly increases assay throughput.
   1. Manual Washing
      1. Label a 50 ml conical tube "1x Wash buffer" and add 45 ml molecular biology grade water and 5 ml 10x Matrix Wash Buffer. Mix buffer well by inversion.
      2. Remove the plate from the rotating plate incubator.
      3. Immediately place the plate on a 96-well magnetic bead separation plate for 5 min.
      4. While keeping the plate on the 96-well magnetic bead separation plate, gently remove and discard the supernatants from the sample wells using a single- or multi-channel pipette. Do not aspirate beads- visually monitor the aspirated buffer in the pipette tip for accidental bead removal. If removal is witnessed, gently add the tip contents back to the well, reseparate, and repeat removal.
      5. Add 300 μl 1x Wash buffer to each sample well.
      6. Fully resuspend the beads by mixing the plate for 30 sec on a microplate mixer.
      7. Incubate the plate on the 96-well magnetic bead separation plate for 2 min.
      8. Remove and discard the supernatants from the sample wells as before.
      9. Repeat steps 6.1.1.5-6.1.1.8 three more times for a total of 4 washes.
      10. With the plate on the 96-well magnetic bead separation plate, visually inspect the wells to confirm even supernatant removal; use a pipette to remove excess residual buffer as necessary. Some buffer will remain in the wells and care must be taken to avoid bead removal or bead drying.
      11. Add 50 μl 1x Reaction buffer to each sample well and mix the plate for 30 sec on a microplate mixer to fully resuspend the beads. If needed, use a pipette to fully resuspend the beads.
   2. Automated Plate Washing
      1. Setup and program the washer according to Table 4. Clean and flush the washer with high-quality (e.g. nanopure) water.
      2. Prime the washer by running the program "Prime".
      3. Remove the plate from the rotating plate incubator.
      4. Immediately place the plate on the 96-well magnetic bead separation plate on the plate washer.
      5. Run the link program "Master Wash". The first program step is a 5 min incubation step where the washer will be stationary.
      6. Following program completion, remove the plate from the washer, add 50 μl 1x Reaction buffer to each sample well, and mix the plate for 30 sec on a microplate mixer to fully resuspend the beads. If needed, use a pipette to fully resuspend the beads.

7. Assay Initiation and Incubation

1. Add 50 μl 0.5 μM A/E reporter (see Section 1) to each sample well and mix for 30 sec on a microplate mixer to fully resuspend the beads.
2. Add 100 μl water to each unused well on the plate to prevent edge effects.
3. Seal the plate with plate sealing tape and incubate the plate using a rotating plate incubator at 750 rpm, 25 °C or RT. Protect the plate from light during incubation.

8. Data Collection and Analysis

Note: This assay is a real-time assay that can be measured multiple times until the desired sensitivity is obtained, there are no stop reagents required. Recommended initial read times are 2, 4, and 24 hr incubation time with assay sensitivity increasing with incubation time.

1. Data collection
   1. At each read time, remove the plate from the rotating plate incubator, remove the sealing tape, and immediately place the plate on the 96-well magnetic bead separation plate. Allow the beads to separate for 2 min.
   2. Place the plate in the microplate reader and measure the emissions at ~470 and ~526 nm under excitation at ~434 nm.
   3. If additional read times are desired, resuspend the beads for 30 sec on the microplate mixer, reseal the plate, and return the plate to the rotating plate incubator.
2. Data analysis
   1. Calculate the emission ratio for each sample by dividing the relative fluorescence unit (RFU) value at 526 nm by the RFU value at 470 nm.
   2. Plot the emission ratio versus the log[BoNT/A] for the standard curve data points. Depending on the BoNT/A potency range tested, a sigmoidal dose-response curve will be obtained (see Representative Results).
   3. Fit the standard curve data with the variable slope dose-response curve Y=Bottom+(Top-Bottom)/(1+10^((logEC₅₀-X)*Hillslope)) where X is the logarithm of concentration, Y is the response, and Y starts at Bottom and goes to Top with a sigmoidal shape.
   4. Determine the limits of detection (LOD), limits of quantification (LOQ), and half-maximal effective concentration (EC₅₀). Limits of detection are defined as a sample having an emission ratio less than 3 standard deviations (SDs) below the blank controls (n = 6). Limits of quantification are defined as a sample having an emission ratio less than 10 SDs below the blank controls (n = 6). EC₅₀ is determined from the sigmoidal dose-response curve fit.
   5. Interpolate the potency of any unknown samples against the sigmoidal dose-response standard curve.
      1. For quantitative results, the unknown sample should ideally fall within the linear portion of the standard curve. Approximate the linear portion of the standard curve by calculating the 20-80% total assay response window (e.g. if the emission ratio of the standard curve ranges from 0.5-2.5, the linear range would be the portion of the standard curve that ranges from 0.9-2.1).
      2. For qualitative results, compare the unknown sample against the LOD and LOQ of the standard curve.
      3. Do not extrapolate unknown samples beyond the limits of the standard curve.

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

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