Bacterial Toxin Research Citations

Induction of active EAE and immunizations:

For EAE induction, mice were immunized subcutaneously with 100 g MOG₃₅₅₅ (CS Bio Co.) emulsified in CFA (made with 5 mg/ml heat-killed Mtb H37Ra [BD] in incomplete Freunds adjuvant [BD]). PTX (List Biological Laboratories) or mutant PTX (mPTX; mutated at two positions in the S1 subunit [R9K and E129A]; List Biological Laboratories) was injected i.p. (300 ng) on days 0 and 2.

Author did not specify which List Labs Pertussis Toxin was utilized.  List Labs provides Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer and Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free).

• Product #184 – Pertussis Toxin Mutant

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.…

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°Chourly 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

Oral immunization:

Aged mice with or without AMSC transfer and young adult mice were immunized three times at weekly intervals with oral doses of 1 mg of OVA (Fraction V; Sigma, St. Louis, MO) and 10 µg of CT (List Biological Laboratories, Campbell, CA) in PBS…

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

Measurement of serum antibody responses of mice and hamsters:

To better standardize the enzyme-linked immunosorbent assay (ELISAs) for consistency, reproducibility, and accuracy for the detection of immune responses to FliC, TcdARBD, and TcdBRBD in mice and hamster serum, we completed a checkerboard dilution series with various concentrations of mouse and hamster serum.³⁷ We coated plates with 10 ng/well, 20 ng/well, 50 ng/well, 100 ng/well, 200 ng/well, and 500 ng/well of commercially available purified TcdA, TcdB (List Biological Laboratories, Campbell, CA, USA) …

• Product #152C – Toxin A from Clostridium difficile
• Product #155 – Toxin B from Clostridium difficile

… The light chain of botulinum neurotoxin type B (50 kDa, List Biological Laboratories, Campbell, CA) was used to cleave recombinant Sb2.13 …

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

Toxin Neutralization Assay:

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

Induction of EAE and Clinical Evaluation:

EAE was induced by subcutaneous injection of 100 µg MOG35-55 peptide (Prospec) emulsified with Complete Freund’s Adjuvant (CFA, 200 µg mycobacterium), followed by intravenous injection of 100 ng pertussis toxin (List Biological Laboratories, Inc.) on day 0 and day 2 post-immunization. …

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)

Induction and 4-MU Treatment of EAE:

All mice were administered 400 ng of pertussis toxin (List Biological) intraperitoneally (i.p.) at 0 and 48 h postimmunization.

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)

Study Animals:

Twelve healthy Quarter Horse foals were used for this study. All foals had age-appropriate results of complete blood count (CBC) on day 2 of life. Individual foals were randomly assigned to a vaccinated group, Group 1 (N = 8), or a control group, Group 2 (N = 4). Group 1 foals received 1 x 10¹¹ CFU of R. equi inactivated by 5 kGy of eBeam irradiation, adjuvanted with 100 µg of the mucosal adjuvant cholera toxin B (CTB, List Biological Laboratories, Campbell, CA, USA), and suspended to a final volume of 100 ml in 0.9% NaCl solution by gavage on days 2, 7, and 14 of life. …

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

Iontophoretic Tracer Injections:

For retrograde tracing, we used a 24% solution of Fluorogold (FG; Fluorochrome) in a sodium acetate buffer in nine animals. In two animals, we injected both FG and 1% cholera toxin subunit B (CTB; List Biological Laboratories) dissolved in distilled water into the IC. …

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

Iontophoretical Injection of PHA-L or CTB:

For injection of tracers, mice were first anesthetized with chloral hydrate at a dose of 400 mg/kg. With the heads of the mice immobilized using a Stoelting stereotaxic apparatus, small holes were drilled through the skulls to form specific injection sites positioned 1.6 mm posterior to bregma, 3.3 mm lateral to midline and 4.2 mm ventral to the dura for the control mice and 1.4 mm posterior to bregma, 3.1 mm lateral to midline and 4.2 mm ventral to the dura for the epileptic mice. 10% CTB (List Biological Laboratories, CA, USA) in distilled water…

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