Bacterial Toxin Research Citations

Anthrax lethal toxin neutralization assay:

Samples collected after release were characterized for toxin-binding activity via an anthrax LeTx neutralization assay that was performed using decreasing amounts of the released samples. Specifically, the biological activity of PANG was measured using a LeTx neutralization assay as described previously with modifications.³⁵ J774A.1 (ATCC TIB-67, Manassas, VA) cells were plated in 96-well flat-bottomed tissue culture plates at 2.5 10⁴ cells/well in 50 L and incubated for 1619 h at 37C with 5% CO₂. Test samples were diluted and titrated on the plated cells in the presence or absence of LeTx (List Biological Laboratories, Campbell, CA). …

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).

Analysis of TMV-PA Antigens for Reactivity against PA Specific Antibodies:

SDS-PAGE, ELISA and Western Blots – 
 
… For westerns, gels were transferred onto 0.2 um nitrocellulose
membranes (7 cm x 8.5 cm) in Tris-Glycine buffer (25 mM Tris, 192 mM Glycine, 20%
Methanol, pH 8.3) for 1 hour at 100V. Membranes were blocked with 2.5% non-fat milk
in TBS (20 mM Tris, 150 mM NaCl, pH 7.6) for 1 hour at room temperature. Primary
antibody (Goat anti-PA polyclonal, List Biologics Lot # 7712A2 …

Cell culture and reagents:

… Protective antigen (PA), lethal factor (LF) and Escherichia coli derived LPS were purchased from List Biological Laboratories Inc. (California USA). The following is a list of antibodies (Abs) used in this study. …

Long-term TIR cells were generated as previously described [110].  Briefly, RAW 264.7 macrophages were treated with a cytolytic dose of LeTx (250 ng/mL LF and 500 ng/mL PA) for 5 h and surviving cells were plated in fresh culture medium.  After two weeks, surviving clones were individually picked and plated on a 96-well plate.  Each clone was tested for LeTx sensitivity and resistant clones were pooled and propagated. Short-term TIR cells were generated as previously described [110].  Briefly, RAW 264.7 macrophages were treated with a sub-lethal dose of LeTx (100 ng/mL LF and 100 ng/mL PA) for 5 h and then supplemented with fresh media overnight.  The next day surviving cells were pooled and plated onto new culture plates with fresh media.  BMDM-TIR cells were generated by Dr. Soon-Duck Ha as previously described [177].  Briefly, BMDMs were treated with a sub-lethal dose of LeTx (100 ng/mL LF and 100 ng/mL PA) for 24 h and surviving cells were plated onto new culture dishes with fresh media. …

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

…Western blot analysis was performed as in [24] with the following modifications. Primary antibodies were diluted in blocking buffer at 1:1000 (Goat anti-PA polyclonal, List Biological Laboratories) or…

Reagents:

Anthrax Lethal Factor and Protective Antigen were 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 #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

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

Reagents and antibodies:

…CT was purchased from List Biological Laboratories (Campbell, CA, USA). …

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

… Stimulation of the NALP1, NLRC4, or AIM2 inflammasome was achieved by treatment for 6 h with 1 g/ml protective antigen (List Biological Laboratories, Campbell, CA, USA) and 1 g/ml anthrax lethal factor (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 #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

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

Chemicals and Reagents:

All toxins were purchased from List Biological Laboratories. …

FRET MAPKKide LF activity:

For drug testing in 96-well plates, the reaction volume was 250l per well, containing 20mM HEPES pH 7.2, 5M MAPKKide conjugated with DABYL and FITC (List Biological Laboratories, Inc), and …

Toxins Treatments:

Cells were seeded in a 96-well plate at a density of 10,000 cells/well 1 d before toxin treatment. Various concentrations of LF or FP59 combined with a fixed concentration of PA (500ng/mL) were added to the wells, and cells were incubated for 6h at 37C. Cell viability was measured by MTT assay. In other toxin treatment experiments, cells were pre-treated with various concentrations of drugs, and then treated with constant concentrations of 8.3g/ml of C. difficile toxin B, 500ng/ml of P. aeruginosa Exotoxin A, or 500ng/ml of Cholera toxin for 6hours. RAW264.7 survival was measured by MTT assay. …

• Product #155 – Toxin B from Clostridium difficile

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

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

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

Recombinant proteins and reagents:

Protective antigen (PA) and edema factor (EF) were purchased from List Biological. …

Cell seeding and incubation with toxins:  

For toxin treatment, patterned and nonpatterned peritoneal macrophages and BMDMs were incubated with PA at 1 g/ml and EF at 100 ng/ml for 6 or 16 h at 37C in a 5% CO₂ atmosphere. For some experiments, cells were incubated with PA alone at 1 g/ml.

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

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

… Recombinant protective antigen of anthrax (PA) and recombinant lethal factor (LF) were purchased from List Biologicals (Campbell, CA, USA). …

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