Bacterial Toxin Research Citations

EAE:

…On D0 and D2 mice were also injected intraperitoneally (ip) with 500 ng pertussis toxin (List Laboratories, Campbell CA) in phosphate-buffered saline (PBS). …

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:

…Two intraperitoneal injections of 182 400 ng pertussis toxin (List Biological Laboratories, Denver, CO, USA) were given 24 and 72 h 183 later. …

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)

Effect of drug treatment on MCH-induced cilia length control:

The following drugs were used in this study: pertussis toxin (PTX; List Biological Laboratories Inc., Campbell, CA); …

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)

…labeling of neurons projecting to the orbitofrontal cortex. These rats received an electrophoretic injection of 0.5% (w/v) cholera toxin B subunit (CTB; #103A; List Biological Laboratories, … Antibody for CTB. The anti-CTB antibody (#703; List Biological Laboratories) …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

… 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)