Bacterial Toxin Research Citations

… 100 l of Pertussis Toxin (5 ng/l) (List Biological Laboratories) was injected intravenously (i.v.) on the day of the immunization and again after 48 hrs. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

EAE induction:

To induce EAE, mice were injected subcutaneously at three locations on day 0 with 200g MOG_35-55 peptide (United Biosystems) emulsified in CFA supplemented with 2mg/ml heat-killed Mycobacterium tuberculosis (VGD, Inc./Voigt Global). On days 0 and 2, mice were also injected intraperitoneally with 200ng of pertussis toxin (List Biologicals). Mice were assessed daily for symptoms and scores were assigned as follows: …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Materials and Methods

Anthrax lethal factor (Product #169), and the chicken IgY polyclonal anti-LF antibody (Product # 769A) are products
of List Biological Laboratories, Inc. The C8 Starwell Maxi Nunc-Immuno Module Plates (cat# 441653) 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 substrate, MAPKKide® Plus, were 1.25 mM in DMSO
based on the peptide content determined by elemental analysis. The substrate was diluted in assay buffer:
20 mM HEPES, pH 8.0 containing 0.1% Tween-20. For the microplate assays, the LF was dissolved in neat bovine
plasma and diluted 1:10 in assay buffer. For the HPLC assays, the LF was added to neat bovine plasma and not
diluted.

LF Activity Assays:

Microplate reader: Assays were performed on a SPECTRAmax GEMINI XS fluorescence microplate reader
(Molecular Devices). The cleavage reaction was initiated by addition of the substrate, MAPKKide® Plus. The
concentration was optimized to minimize background fluorescence while maintaining measureable cleavage. For
all experiments the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 or 6 hours followed
by an additional 18 to 18.5 hr overnight incubation at ambient temperature. The excitation wavelength was set to
368 nm and emission to 452 nm with a cutoff filter at 435 nm.

HPLC: The C8 Starwell Maxi Nunc-Immuno Module Plates were coated with 150 μl of a 10 μg/ml solution of
a chicken affinity purified polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A). Plates were incubated
with the IgY overnight at 2-8°C and washed three times with 0.1M Glycine-HCl, pH 2.5. This wash was included to
liberate residual LF retained after the affinity purification of the antibody and was necessary to minimize the background. After 6 washes with PBS containing 0.05% TWEEN-20 (PBST), the anti-LF coated wells were exposed to
300 μl of a series of LF concentrations in neat plasma. The plates were incubated at 22°C for 2 hours. Plates were
then washed 6 times with PBST and 250 μl of 1.25 μM MAPKKide® Plus was added. The reaction was allowed to
proceed for 2, 3.5, and 5 hours at 37°C and overnight at ambient temperature. At each time point 200 μl of the
reaction mixture was removed from replicate wells and placed in HPLC sample vials.

HPLC was performed using a Zorbax Eclipse Plus C18 reverse phase column, 4.6 x 150 mm (Agilent) and a guard
column containing the same resin in a Varian ProStar HPLC system (Agilent). Solvent A was 0.1% TFA in water and
solvent B was 0.1% TFA in acetonitrile. The 16 minute HPLC method was as follows: 25% B for 0.75 minutes;
25 to 45% B in 4.75 minutes; 45 to 100% B in 0.75 minutes; 100% B for 3.75 minutes; 100 to 25%B in 0.67 minutes
and 5.34 minute equilibration with 25% B. The column effluent was monitored using an Hitachi fluorescence detector
with excitation set to 350 nm and emission at 450 nm to detect the free coumarin fluorophore cleaved from
MAPKKide® Plus. The injection volume was 20 µl. The 7-amido-4-methylcoumarin (AMC) peak retention time
was 4.8 minutes.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #769B – Anti-Lethal Factor from B. anthracis (Chicken lgY)
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

Materials and Methods

Anthrax lethal factor (Product #169), and the chicken IgY polyclonal anti-LF antibody (Product # 769A) are products
of List Biological Laboratories, Inc. The C8 Starwell Maxi Nunc-Immuno Module Plates (cat# 441653) 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) and sheep plasma (cat# 7319006) were purchased from Lampire Biological
Laboratories. The milk (2% Lucerne) came from the local market.

Sample Preparation: Stock solutions of the fluorogenic substrate, MAPKKide® Plus, were 1.25 mM in DMSO
based on the peptide content determined by elemental analysis. The substrate was diluted in assay buffer:
20 mM HEPES, pH 8.0 containing 0.1% Tween-20. For the microplate assays, the LF was dissolved in neat bovine
plasma and diluted 1:10 in assay buffer. For the HPLC assays, the LF was added to neat bovine, sheep plasma or
2% milk without dilution.

LF Activity Assays:
Microplate reader: Assays were performed on a SPECTRAmax GEMINI XS fluorescence microplate reader
(Molecular Devices). The cleavage reaction was initiated by addition of the substrate, MAPKKide® Plus. The
concentration was optimized to minimize background fluorescence while maintaining measureable cleavage. For
all experiments the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 or 6 hours followed
by an additional 18 to 18.5 hr overnight incubation at ambient temperature. The excitation wavelength was set to
368 nm and emission to 452 nm with a cutoff filter at 435 nm.

HPLC: The C8 Starwell Maxi Nunc-Immuno Module Plates were coated with 150 μl of a 10 μg/ml solution of
a chicken affinity purified polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A). Plates were incubated
with the IgY overnight at 2-8°C and washed three times with 0.1M Glycine-HCl, pH 2.5. This wash was included to
liberate residual LF retained after the affinity purification of the antibody and was necessary to minimize the background. After 6 washes with PBS containing 0.05% TWEEN-20 (PBST), the anti-LF coated wells were exposed to
300 μl of a series of LF concentrations in neat plasma. The plates were incubated at 22°C for 2 hours. Plates were
then washed 6 times with PBST and 250 μl of 1.25 μM MAPKKide® Plus was added. The reaction was allowed to
proceed for 2, 3.5, and 5 hours at 37°C and overnight at ambient temperature. At each time point 200 μl of the
reaction mixture was removed from replicate wells and placed in HPLC sample vials.

HPLC was performed using a Zorbax Eclipse Plus C18 reverse phase column, 4.6 x 150 mm (Agilent) and a guard
column containing the same resin in a Varian ProStar HPLC system (Agilent). Solvent A was 0.1% TFA in water and
solvent B was 0.1% TFA in acetonitrile. The 16 minute HPLC method was as follows: 25% B for 0.75 minutes;
25 to 45% B in 4.75 minutes; 45 to 100% B in 0.75 minutes; 100% B for 3.75 minutes; 100 to 25%B in 0.67 minutes
and 5.34 minute equilibration with 25% B. The column effluent was monitored using an Hitachi fluorescence detector
with excitation set to 350 nm and emission at 450 nm to detect the free fluorophore cleaved from MAPKKide® Plus.
The injection volume was 20 µl. The 7-amido-4-methylcoumarin (AMC) peak retention time was 4.8 minutes.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #769B – Anti-Lethal Factor from B. anthracis (Chicken lgY)
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

Materials:

…Botulinum toxin-B List biological Cat# 620A …

• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant

…On day 0, mice received an additional 500 ng of pertussis toxin ip (List Biologicals) ( Cihakova et al., 2004 ; Smith, 2005). 2.3. Adoptive transfer of EAM. For active adoptive transfer of EAM, male …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Calcium imaging:

…After imaging for approx. 5 min. to establish baseline conditions, ligands were superfused onto the cells as indicated in figures using a gravity-driven perfusion system. For some experiments, cells were pretreated with pharmacological substances (H89, KT5720, BIM, mSIRK or mSIRK-L9A, Figure 6 and Figure 7 figure supplement 3), for 30 min. by adding them to the culture medium during Fura2AM loading. Pertussis toxin (PTX, 100 ng / ml, List Biological Laboratories, Campbell, CA, USA) was added to the culture medium in the incubator for 16 24 h and washed off the extracellular medium before loading with Fura2-AM. … 

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

…Mice received 150 ng pertussis toxin (List Biological Laboratories) intraperitoneally on days 0 and 2. Passive-transfer EAE was induced by injection of 2D2 TH17-polarized cells (as described above) intravenously into recipient mice at 5 106 to 7.5 106 cells/mouse. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

For the induction of peanut extract (PE) -induced food allergy, C57BL/6 mice were sensitized orally once a week for four weeks with 2 mg PE adjuvanted with 20 µg cholera toxin (List Biologicals 101B) followed by either four oral challenges on four consecutive days with 10 mg PE (in this model, symptoms were scored after the first three challenges) or two i.p. challenges (with a two -day break) with 1 mg PE (in that case, symptoms were scored after the first challenge). …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Neutralizing Antibody Assay:

Toxin neutralizing properties of antiserum were determined using Vero cells and C. difficile purified toxin B (List Biological Labs, Campbell, CA, United States).

• Product #155 – Toxin B from Clostridium difficile