The overall goal of this procedure is to quantify the proteolytic activity of botulinum neurotoxin in complex matrices such as pharmaceutical, environmental, and food samples. This is accomplished by first processing test and standard curve samples if necessary, to establish suitable pH and viscosity conditions. Next, the botulinum neurotoxin is immuno precipitated from the samples using antibody coated magnetic beads.
Then the magnetic beads are thoroughly washed to remove any interfering compounds and resuspended in an optimized reaction buffer. Finally, the washed beads are incubated with a protein reporter and spectroscopically measure the cleavage of the reporter over time. Ultimately, comparison of reporter cleavage in the test samples versus the standard curve samples is used to quantify the activity of the botulinum toxin in the test samples with mouse to near mouse bioassay sensitivity.
The main advantages of this technique over other methods such as the mouse, bioassay, immunologic, and other fluorescent methods are that this assay does not require the use of animals and has been demonstrated for use in highly complex matrices found in food, environmental, and pharmaceutical samples. Generally, individuals new to this method may struggle because careful sample processing and dilution is required. Demonstrating this procedure will be Dr.Mark Dunning, a scientist at Bio Sentinel.
Prepare buffers according to the text protocol to generate a standard curve for quantifying test samples, weigh out 10 plus or minus 0.01 grams of solid food sample into a 50 milliliter conical tube and set aside this sample will be used as the diluent in a second tube weigh out two plus or minus 0.01 grams of solid food sample. Add botulinum neurotoxin or bought to the surface of the two gram food sample to a final concentration of 30, 000 MLD 50 per gram of food. Incubate the diluent and spiked samples at room temperature or four degrees Celsius for two hours to give the bot time to interact with the food matrix.
Mimicking a natural contamination, refer to the text protocol for additional sample types. Next, add one milliliter of GPB per gram of food to the spiked and uns spiked samples, and use a pestle to homogenize until thoroughly blended. Extrapolate the total volume of the two gram bot a spiked sample from the approximate total volume of the 10 gram diluent sample.
Then add a one 10th volume of 10 x neutralization buffer to the 10 gram diluent sample and two gram bought a spiked sample based on their total volumes. Mixed samples well by inversion, partially clarify both samples by centrifuging for 10 minutes at 6, 000 times gravity and four degrees Celsius. Then immediately remove the supernatants and transfer to new tubes using the bot a spiked sample as D one and the nons spiked sample as diluent generate the remaining standard curve samples in 1.5 milliliter micro centrifuge tubes.
To prepare solid food samples begin by weighing out two plus or minus 0.01 grams of solid unknown sample into a 50 milliliter conical tube. Add two milliliters of GPB and use a pestle to homogenize the sample. Next, add one 10th the volume of 10 x neutralization buffer to the sample and mix well by inversion after partially clarifying the sample by centrifugation for 10 minutes at 6, 000 times gravity and four degrees Celsius immediately transfer at least 750 microliters of supernatant to a micro centrifuge tube.
This is unknown dilution one. Add 675 microliters of the diluent to two tubes labeled unknown dilution two and three. The diluent will be the same processed material used for standard curve generation.
Use dilution one to make serial dilution by transferring 75 microliters of dilution one into the dilution two tube and mixing. Then transfer 75 microliters of dilution two into the dilution three tube and mix. To clarify the samples, centrifuge them for five minutes at at least 14, 000 times.Gravity.
Immediately remove the supernatants and transfer to new tubes to set up a plate for samples, add 20 microliters of 10 x binding buffer to each well each unknown sample and the standard curved samples D one through D eight require three wells and sample D nine requires six wells. Add 200 microliters of each sample to the respective wells and use a microplate mixer to mix the plate for 10 seconds. Vortex the IPA beads for 10 seconds at the highest speed or until fully resuspended.
Then pipette 20 microliters of the beads into each sample well and mix the plate for 30 seconds. Incubate the plate in a rotating plate incubator for two hours at 750 RPM and 25 degrees Celsius or room temperature to wash the plate using an automated plate washer. After running the prime program, place the plate on the 96 well magnetic bead separation plate on the plate washer.
Then run the master wash program. When the program is complete, remove the plate from the washer. Add 50 microliters of one x reaction buffer to each sample well and mix for 30 seconds.
To initiate the bot test matrix assay, add 50 microliters of 0.5 micromolar AE reporter. To each sample well to prevent edge effects, add 100 microliters of water to each unused. Well use ceiling tape to seal the plate, shield it from light and incubate it at 750 RPM and 25 degrees Celsius or room temperature.
At each read time, remove the plate from the incubator. Remove the ceiling tape, and immediately place the plate On the 96 well magnetic bead separation plate, allow the beads to separate for two minutes. Place the plate in the microplate reader and measure the emissions at about 470 and about 526 nanometers under excitation at about 434 nanometers.
If additional read times are desired, after resus suspending the beads for 30 seconds on the microplate mixer, reseal the plate and return it to the incubator. Shown here are assay results Using bot a holo toin spiked into PBS and tested following two, four and 24 hour incubations with the AE reporter. Cleavage of the reporter by bot is measured as a reduction in the emission ratio measured by our plate reader as approximately 2.7 for the intact reporter to about 0.7 for the fully cleaved reporter.
Specific values will be different between plate readers as seen by the leftward shift in the curve, prolonged incubation time with the reporter results in increased reporter cleavage. The data points tested in triplicate display low standard deviation of the mean and follow the expected trend of increased emission ratio with decreased toxin load. Failure of the assay to follow this expected trend may indicate an error during dilution generation or data plotting the emission ratios of the controls containing no bot A also remain steady during the incubation indicating the lack of non-specific protease activity.
This figure demonstrates the general methodology used to detect or quantify any unknown sample against a standard curve and quantifies pharmaceutical bot a samples. A standard curve was generated in PBS with purified bot, a holo toin and processed in parallel with dilutions of drug product generated from a single 100 unit vial of lyophilized Botox rehydrated in 0.9%saline in this assay, the linear portion of the standard curve falls between an emission ratio of 2.11 and 1.05, and is indicated by the dashed box in the figure. The concentrations of the three unknowns that fall within this linear range were then interpolated from the standard curve.
In this experiment, fresh tomatoes and 2%milk were used as solid and liquid food matrices and spiked with bot a complex comprised of the core hollo, toin, and neurotoxin associated proteins. This combination was selected because it resembles the toxin produced during a natural clostridium contamination as shown here. Recovery of bot A is observed with both matrices.
Furthermore, increased incubation time with the reporter increases assay sensitivity but does not result in decreased emission ratios for the no toxin controls indicating the observed cleavage results from bot A and not non-specific protease carry over from the food in the assay. The bot A-L-O-D-L-O-Q and EC 50 for both foods at each time point shown are summarized in this table. The LOD and LOQ are defined as the lowest concentration sample with an emission ratio lower than three and 10 standard deviations below background respectively.
Some matrix to matrix variability in LOD and LOQ is expected as matrix effects may influence the binding of the toxin to the beads and the recovery of beads during washing. While it would appear that there was more toxin recovery from 2%milk than tomatoes from the data, much of this difference results from the additional dilution required to homogenize tomato samples in GPB. In addition to being a solid food matrix, tomatoes are a noteworthy sample type where pH and ionic strength adjustment is critical to assay success.
This figure illustrates assay responses when testing tomatoes with or without inclusion of the 10 x neutralization buffer. Failure to add the buffer results in poor recovery of bot A from samples and is demonstrated by a constant emission ratio across all tested bot A concentrations addition of the 10 x neutralization buffer, however, results in sensitive toxin detection, nonspecific proteases may be endogenous to the food sample or introduced when using non purified bot preparations such as Clostridium culture, supernatants, and may cleave the AE reporter leading to false positive results. This example demonstrates nonspecific protease activity found in Clostridium bot a culture supernatants using a modified reporter that is no longer cleaved by bot A.The high levels of reporter cleavage indicate the culture.
Supernatant contains significant protease activity, which is effectively negated by the addition of protease inhibitors. Once mastered, this technique can be performed in between four and 26 hours total assay time, depending on sample type and toxin load. After watching this video, you should have a good understanding of how to isolate and quantify botulinum neurotoxin from complex samples using immunoprecipitation in a fluorescence based protein reporter system.
Don't forget that working with botulinum neurotoxins can be extremely hazardous and precautions such as gloves, laboratory codes, and chemical and biological safety cabinets should be used while performing this procedure.
