Bacterial Toxin Research Citations

… Activation of the NLRP1 inflammasome was achieved by treatment for 16 h with anthrax lethal toxin (2.5 lg/ml Anthrax Protective Antigen and 1 lg/ml Anthrax Lethal Factor mix, List Biological Laboratories, 171D and 169A). …

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).

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

Vaccine preparation and immunization:

DPX-rPA and alum-rPA were provided by IMV Inc. (Halifax, Canada), but AVA was provided by the CDC. Test and vehicle/control article formulations were prepared fresh on the day of dosing in accordance with the method provided. DPX-rPA was prepared as previously described¹⁹ containing a poly I:C (Thermo Fisher) or Pam3CSK4 (EMC Microcollections) adjuvant and a 10:1 mixture of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine):cholesterol (Lipoid GmBH, Germany) or S100:cholesterol (Lipoid GmBH), see Supplementary Table 1 for complete formulation details for each figure. The source of rPA was DRDC, List Biologicals, or Pfenex Inc.²⁶ …

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

… B. anthracis LF was purchased from List Biological Laboratories. …

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).

Reagents:

… B. anthracis lethal factor (LF) was acquired from List Biologicals (#172C). …

J774.A1 culture and stimulation:

J774.A1 cells were cultured in IMDM (Lonza) containing 10% (vol/vol) heat-inactivated FBS, 1% (vol/vol) nonessential amino acids (Lonza), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37C in a humidified atmosphere containing 5% CO₂. Cells were scraped for passages and, for experiments, counted and seeded into 96-well plates in culturing medium. On the next day, cells were changed to fresh media and stimulated with vehicle or 1 g/ml PA combined with 0.5 g/ml LF (LeTx), VBP (0.1% TFA in DMSO) or 1G244 (DMSO) at a final concentration of 10 M or 25 M.

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

EXPERIMENTAL –  FRET Lethal Factor Protease Assay:

The FRET assay measures the enzymatic activity of LF with an internally quenched peptide substrate derived from MAPKK. LF cleavage of the substrate separates the quencher and fluorescent tag leading to an increase in fluorescence intensity. The slope of fluorescence absorbance is therefore used to determine enzyme activity. Anthrax lethal factor (30 nM final concentration) and MAPKKide substrate (25 M final concentration) were purchased from List Biological Laboratories, Campbell, CA. …

Author did not specify which MAPKKide substrate was utilized; List Labs provides the following:

• 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

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).

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

… Recombinant anthrax lethal factor was purchased from List Biological Laboratories, Inc. …

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).

Test approach:

No standardized approaches are available to investigate anthrax seroprevalence in wildlife. Thus, samples were tested for antibodies against the anthrax protective antigen (PA, List Biological Laboratories Inc) using an in-house ELISA and an in-house Western Blot and for antibodies against anthrax lethal factor (LF, List Biological Laboratories Inc) using an in-house Western Blot. …

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).

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