Bacterial Toxin Research Citations

Protease assay:

Proteolytic activity was determined, as described earlier [22Kaman WE, Hulst AG, van Alphen PT, et al. Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillusspecies. Anal Chem. 2011;83:25112517.[Crossref], [PubMed], [Web of Science ], [Google Scholar]], by incubating 16 M FRET-peptide with varying concentrations of PA₈₃ or LF (both List Laboratories) in HEPES buffer (20 mM HEPES, 0.05% Tween-20, pH 8.2) using blackwell clear bottom 96-well plates (Corning). …

Cleavage characteristics of PA₈₃

20 g/mL PA₈₃ was incubated with 16 M FRET-peptide PEK-054 (FITC-NleKKKKVLPIQLNAATDK-KDbc) [20Cummings RT, Salowe SP, Cunningham BR, et al. A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease. …

LC MS/MS:

Prior to LC MS/MS analysis 10 g PA₈₃ was digested with 0.5 g trypsin (Difco, BD Biosciences) for 3 hr at 37C. The trypsinized sample was analyzed by MS/MS using the data dependent LC-auto MS/MS mode. …

SDS-PAGE:

PA₈₃ protein (2 g) was mixed 1:1 with 2x Laemmli sample buffer (Biorad), incubated for 5 min at 95C and loaded onto a 10% SDS-PAGE gel. After electrophoresis for 1 hr at 22 mA (max. 160 V) the gel was stained overnight with PageBlue staining solution (Fisher Scientific) and washed two times to visualize bands.

Specificity testing PA₈₃

20 g/mL PA₈₃ was incubated with 15 g/mL B. anthracis protective antigen antibody-[HRP] (-PA, Novusbio, BAP0102), 15 g/mL human IgG antibody-[HRP] (-human IgG, Sigma Aldrich, A0170) or PBS (solvent of the used antibodies) in HEPES buffer for 5 hr at room temperature. Next, 1 L PEK-054 (800 M) was added to the reactions and increase in fluorescence was measured at 37C for 60 min as described above.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

REAGENTS AND SOLUTIONS:

Anthrax protective antigen and anthrax lethal factor, 1 mg/ml

Dissolve anthrax lethal factor and anthrax protective antigen (List Biological Labo-

ratories, cat. no. 172B and 171E) each to a nal concentration of 1 mg/ml in sterile

dH2O. Divide into 10-l aliquots or an appropriate volume for the users experiments

and store up to 2 years at 80C. Avoid freeze-thaw cycles. Remove one aliquot of

each from the freezer 30 min prior to starting the experiment. Allow them to come to

room temperature. Enzymatically inactive anthrax lethal factor (List Biological Lab-

oratories, cat. no. 176) can be used as a negative control, if desired. Good biosafety

level 2 laboratory practices should be employed in the safe handling of this reagent.

Protective antigen and lethal factor should be stored separately.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #176 – Anthrax Lethal Factor (LF), Recombinant, Mutant E687C from B. anthracis

LPS, 1 mg/ml

Prepare a 1 mg/ml stock of Salmonella Minnesota R595 (Re) LPS (e.g., List Bi-

ologicals, cat. no. 434) in H2O containing 0.5% triethylamine. Divide into 100-l

aliquots and store up to 1 year at 4C.

• Product #434 – ULTRA PURE LPS from Salmonella minnesota R595 (Re)

Flow cytometry:

Cells were trypsinized, rinsed in cold PBS-BSA, and labeled with m825-MMAE, an isotype-matched human IgG, or FITC-conjugated protective antigen (List Biologicals Laboratories) in PBS-BSA at 4^o C. …

• Product #175 – Anthrax PA 63 – FITC Conjugate

In vivo studies to determine PA removal from rat blood:

… PA (List Biological Laboratories, US) was administered by intraperitoneal (IP) injection (100g/kg) or infused via the venous catheter for 1hour at 100g/kg. In the filter studies, PA was infused as before for 1hour prior to connection of the extracorporeal circuit and continued at 40g/kg for the remaining 2hours of the study. After the initial 1-hour infusion period, the arterial catheter was connected to either a Valortim antibody bound or blank control cryogel columns (5cm by 1cm (length x diameter)) via a peristaltic pump. …

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Reagents and Antibodies:

… Anthrax lethal toxin was purchased from List Biological Laboratories, …

LDH cytotoxicity assays:

RAW 264.7 cells were seeded at 0.5 106 cells/well in 6-well plates in 15 
standard growth medium 24 hours prior to treatment. Cells were treated with the indicated agents
for the indicated times. Supernatants were then harvested and analyzed for LDH activity using an
LDH cytotoxicity assay kit (Pierce).  For experiments with proteasome inhibitors or bestatin
methyl ester, cells were treated with these agents 30 minutes prior to the addition of Val-boroPro
and/or LT, and then incubated for the indicated amount of time. …

Immunoblotting experiments:

RAW 264.7 macrophages were seeded at 0.5 106 cells/well in
6-well plates or at 5 106 cells/dish in 10 cm plates, and 24 h later were treated with agents as
described. Bestatin methyl ester and bortezomib, if used, were added 30 minutes prior to LT …

Immunoprecipitation experiments:

For immunoprecipitation experiments, HEK 293T cells were
seeded at 7 106 cells/well in 10 cm plates and transiently transfected with 5 g of N-terminal V5
and C-terminal FLAG-tagged Nlrp1b and 5 g of N-terminal HA-tagged ubiquitin using Fugene 10 
HD. After 24 h, cells were then treated with bortezomib for 30 minutes followed by LT or vehicle
for an additional 3 hours and harvested. …

Anthrax Lethal Toxin (LT) = Anthrax Lethal Factor (LF) + Anthrax Protective Antigen (PA)

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

… Anthrax lethal factor and edema factor were from List Biological Laboratories (Campbell, CA). …

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence)

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Reagents:

Recombinant expression and purification of LFn-FlaA was performed as previously described [19]. B. anthracis protective antigen (PA) and lethal factor (LF) were acquired from List Biologicals. …

Macrophage differentiation and stimulation:

…  For NLRP1b inflammasome activation, cells were stimulated with LeTx (1g/ml PA combined with 0.5 or 1g/ml LF). …

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Materials and Methods

Anthrax lethal factor (Product #172), and the chicken IgY polyclonal anti-LF antibody (Product # 769A) are
products of List Biological Laboratories, Inc. The Nunc-Immuno Tubes, Maxisorp (cat# 444202) used for LF
antibody coating and the dimethyl sulfoxide (DMSO) (cat # TS-20684) were purchased from ThermoScientific.
The 96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning
(cat # 3991). Bovine plasma (cat # 7310806) was purchased from Lampire Biological Laboratories.

Sample Preparation: Stock solutions of the fluorogenic substrates, MAPKKide® Plus (± biotin), were made
2.5 mM in DMSO based on the peptide content determined by elemental analysis. The substrates were diluted
in assay buffer: 20 mM HEPES, pH 8.0 containing 0.1% Tween-20. The LF was added to neat bovine plasma.

LF Activity Assays:
Antibody Capture: The Nunc-Immuno Maxisorp Tubes were coated with 1 mL of a 10 μg/ml solution of
a chicken affinity purified polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A).
The immuno-tubes were incubated with the IgY overnight at 2-8°C. After 5 washes with 1.5 mL each of PBS containing 0.05%
TWEEN-20 (PBST), the anti-LF coated tubes were exposed to 1 mL of a series of LF concentrations in neat
plasma. The tubes were incubated at 22°C for 1 hour. Tubes were then washed 3 times with 1.5 ml PBST and
1.0 ml of 10 μM MAPKKide® Plus (± biotin) was added. The reaction was allowed to proceed for 3, 5 and
22 hours at 37°C. At each time point 250 μl of the reaction mixture was removed from triplicate tubes and placed
in a 96 -well black plate. In the case of the biotinylated MAPKKide® Plus a 0.5 ml aliquot of each triplicate
was added to streptavidin-coated beads after 22 hours of exposure to LF. The mixture was gently rotated at
ambient room temperature for 1 hour and then read in the platereader.

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #769B – Anti-Lethal Factor from B. anthracis (Chicken lgY)
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

… MAPKKide was obtained from List Biological Laboratories (Campbell, CA 

Author did not specify which List Labs MAPKKide was utilized; the following are available:

• Product #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor
• Product #531 – MAPKKide Peptide Substrate (DABCYL/FITC) for Anthrax Lethal Factor
• Product #532 – MAPKKide Plus (AMC) Specific Substrate for Anthrax Lethal Factor

Detection of Toxin Agents:

… As illustrated in Figure 4a, the derived GT1b LSPR chip utilized Au nanoparticles of 40 nm in size. In the present study, a heavy chain binding domain (BTX/A/Hc) was used instead of the neurotoxins (BTX/A) composed of Hc and Lc with potent toxicity.  To assess the LSPR biosensor unit, we examined the potential utility of the biosensor using different concentrations of RCA120, ricin (RCA60) and BTX/A/Hc in solution.

• Product #612A – Botulinum Neurotoxin Type A Heavy Chain, Recombinant, Binding Domain