Bacterial Toxin Research Citations

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

… 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

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