Bacterial Toxin Research Citations

Reagents:

… CTB (choleragenoid, 103B) and Clostridium difficle toxin B (TcdB) (6246-GT-020) were purchased from List Biological Laboratories and R&D Systems, respectively. …

Measurement of IL-1 production in vitro:

For measuring IL-1 production in vitro, 2 10⁵ cells per well in 96-well plates were first cultured for 5 h in the absence or presence of LPS O55:B5 or LPS O111:B4. Then CTB, ATP, R848 or TcdB was added at indicated concentrations and further cultured for indicated hours. A pan-caspase inhibitor, Z-VAD-FMK (4800-520, MBL), and a protein kinase A (PKA) inhibitor, H-89 (10010556, Cayman Chemical), were added 3 h before addition of CTB or ATP. Dimethyl sulfoxide (DMSO, Nacalai Tesque) was used as a solvent control for inhibitors. …

Measurement of IL-1 production in vivo:

Mice were injected intra-peritoneally (i.p.) with 100 g LPS O55:B5 per head. Five hours later, CTB was injected i.p. at 100 µg per head. PBS was used as a control for CTB. Then further 6 h later, peritoneal lavage fluids were harvested and subjected to ELISA for IL-1.

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

Assessment of toxin neutralizing antibodies

Toxin neutralization assay was performed as previously described^4,5,6. Briefly, sample dilutions were added to J774 macrophages cultured in cultured in RPMI supplemented with 10% fetal calf serum. Bacillus anthracis lethal toxin (LeTx) [i.e., PA plus Bacillus anthracis lethal factor (LF, List Biological, Campbell, CA)] was then added to the plates. After overnight incubation, MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was added to assess the viability of macrophages as a function of redox potential. Toxin neutralizing antibody titers were determined as the lowest concentrations of sera  that protect macrophages from the cytotoxicity of LeTx.

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 #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

… Pertussin Toxin List Biological Laboratory Cat. #181 …

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)

… Pertussis toxin List Biological Laboratories Cat#180 …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

… To induce EAE, adult female mice were injected subcutaneously in the flank/tail base with 200 µg MOG 3555 peptide emulsified in complete Freunds adjuvant (BD Biosciences) supplemented with 600µg Mycobacterium tuberculosis (strain H37Ra; BD Biosciences). Two i.p. injections of 400 ng pertussis toxin (List Biological Laboratories) were given 0 and 48 hours later. Clinical scores (0, healthy; 1, flaccid tail; 2, ataxia and/or paresis of hind limbs; 3, paralysis of hind limbs and/or paresis of forelimbs; 4, tetraparalysis; 5, moribund or death) were recorded daily …

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)

Diphtheria– and tetanus-specific MBC detection by flow cytometry:

Protein biotinylation and formation of Agquantum dot (QD) complexes was performed using diphtheria toxin CRM197 (DT; List Biological Labs, CA), tetanus toxoid (TT; List Biological Labs), and human serum albumin (HSA; Sigma-Aldrich; used as a negative control) that were biotinylated using a ChromaLink Biotin Kit (Solulink, CA) according to the manufacturers instructions. …

• Product #149A – Diphtheria Toxin CRM₁₉₇, Mutant
• Product #191A – Tetanus Toxoid from Clostridium tetani

Food allergy model:

Peanut butter (3.75 mg; ~1 mg of protein; Kraft, Northfield, IL) with 10 µg of cholera toxin (List Biologicals, Campbell, CA) in 0.5 mL of PBS was administered intragastrically (Delvo SA, Biel, Switzerland) weekly for 4 weeks. …

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

Antibodies:

Primary antibodies used in this study that are commercially available include: rabbit anti-MEK2 (Santa Cruz Biotechnology sc-523, AB_2281672), goat antiprotective antigen from Bacillus anthracis(List Biological Laboratories #771B), …

LFNEffector intoxication of T84 cells:

… For intoxication of cells, media was replaced over 105 cells previously seeded in a 12-well plate. 31.7 nM PA alone (List Labs, #171E) or in the presence of 13.6nM LFN, LFN-ACD, or LFN-RID was added to media and cells were incubated for 20 hr at 37C in the presence of 5% CO2. For co-intoxication of 13.6 nM LFNACD and LFN-RID, 3x excess PA was used to ensure equal translocation of both effectors.

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

… For the induction of MP4-EAE, mice received 200 µg MP4 (Alexion Pharmaceuticals Inc) emulsified in CFA and 200 ng pertussis toxin (List Biological Laboratories, #181, Lot #18123611) in 500 µl sterile phosphate-buffered saline (PBS) on the day of immunization …

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)