Bacterial Toxin Research Citations

BA spores and antigens:

… BA PA and LF were purchased from List Biological Laboratories, Inc. (Campbell, CA, USA).

Primary and secondary T-cell responses to BA:

The in vitro human T-cell assay was adapted from standard assays conducted for the study of human immune responses… To assess secondary recall T-cell responses to BA, cells from the 7-day co-culture were harvested, washed, counted, and replated at a hDC:TC ratio of 1:10 in 24-well plates for 48 h with a second preparation of autologous hDCs that were infected with BA spores (WT or gerh) at a MOI of 1:1, 2.5 g/mL PA, 1 g/mL LF, KLH (10 g/mL), or left untreated.

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:

Lyophilized recombinant PA and LF were purchased from the List Biological Laboratories, Inc. (Campbell, CA), and reconstituted in sterile water to make stock solutions with final concentrations of 1 mg/ml.

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

Gene Expression Profiling:

RNA was isolated from HepG2 cells cultured under normoxic or hypoxic conditions with or without LT using TRIzol according to…

Polysome Analysis:

Polysome analysis was performed as described previously (37). In brief, HepG2 cells were pretreated with or without LT for 3 h and then cultured under hypoxic conditions for 4 h. …

Cytotoxicity Assay:

HepG2 and Hepa1c1c7 cells were cultured in 96-well plates with or without LT either 3 h prior to or at the initiation of a 24-h incubation under hypoxic conditions. …

Immunizations and serum isolation:

Groups of eighteen (18) female C57Bl/6 (National Cancer Institute/Charles River Laboratories, Wilmington, MA) mice at 8-12 weeks of age were subcutaneously immunized with saline, 5 g recombinant anthrax protective antigen (rPA; List Biological Laboratories, Inc., Campbell, CA) alone or with 1.3 mg alum (Alhydrogel; Sigma, St. Louis, MO). On day 71 post-immunization, three mice from each group were given a boost of rPA (no adjuvant) at the same dose as the primary immunization …

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

… A 50 L of anthrax toxin PA63 (List Biological Labs, Campbell, CA, USA) dissolved in 50 mM carbonatebicarbonate buffer (pH 9.6) was used to coat each well of a 96-well plate…

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

Intracellular detection of LFn fusion proteins and PARP cleavage:

… Detection of toxin proteins was achieved with rabbit anti-LF and mouse anti-CdtB, both made in our laboratory, and goat anti-Pseudomonas exotoxin A (List Biological Laboratories, Campbell, CA, USA). …

• Product #760 – Anti-Exotoxin A (Goat) from Pseudomonas aeruginosa

Anthrax toxin neutralization assay (TNA):

The anthrax TNA was performed using the mouse macrophage cell line J774A.1 (European collection of cell cultures, ECACC) as previously described [25] with slight modifications. 96-well flat-bottomed tissue culture plates (Greiner Bio One, Germany) containing 810⁴ macrophages/well in DMEM (Life Technologies, USA) and 10% FCS (Life Technologies, USA) were incubated overnight at 37C and 5% CO₂. Goat sera were doubly diluted (1:50 to 1:6400) in culture medium containing PA and LF (List Biological Laboratories Inc., Campbell, CA) at concentration of 500 ng/ml and 400 ng/ml (lethal toxin, LT) respectively. The sera/LT mixture was incubated for one hour at 37C and 5% CO₂ before adding to overnight cultured cells (after discarding medium) and incubated for three hours. …

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

Materials:

B. anthracis PA protein was from List Biological Laboratories, Inc. (Campbell, CA).

Adsorption of protein antigens on aluminum hydroxide particles:

The adsorption of proteins (OVA or PA) on aluminum hydroxide particles was carried out by mixing the particles in suspension with the protein in solution. Briefly, a certain volume of the protein solution was added into a tube (10 g OVA or 4 g PA), followed by the addition of particles in suspension at a weight ratio of 1:5 to 1:1 (OVA vs. particles) or 1:5 (PA vs. particles). After 20 min of gentle stirring, the protein-particle mixtures were stored at 4C or freeze-dried, if needed, before further use.

Immunization studies:

…When PA was used as the antigen, female BALB/c mice (1820 g) were immunized subcutaneously with PA-adsorbed aluminum hydroxide particles on days 0 and 14. As negative controls, mice were injected with sterile PBS or PA alone. The dose of PA was 4 g per mouse per injection, and the dose of the particles was 20 g per mouse per injection.

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

Reagents and Materials:

… Protective antigen (PA) protein of Bacillus anthracis was purchased from List Biological Laboratories, Inc. (Campbell, CA; USA). …

Rapid Peptide Specificity Analysis:

The peptide specificity analysis was performed by CE using PA (positive control), BSA, HRP, and an anti-PA mAb at 20 kV as described above. …

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

In vitro protease assays:

Purified InhA1 was mixed with the substrate in protease buffer (50 mM Tris, 100 mM NaCl, 1 mM CaCl₂, 1 mM MgSO₄ [pH 7.8]) and incubated at 37C for set time points. Reaction mixtures were then placed on ice, and EDTA was added to a final concentration of 50 mM to stop the reaction. SDS buffer (2) was added, and samples were boiled for 5 min, separated by SDS-PAGE, and analyzed by Western blotting with substrate-specific antibodies. Recombinant PA (rPA), rLF, and rSodA-1 from B. anthracis were obtained from BEI Resources (ATCC, Manassas, VA). rEF was obtained from List Biological Laboratories (Campbell, CA). rNpr599 was purified as described above.

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).

Supernatant ELISAs and LDH assays:

…Cells were infected at a multiplicity of 3:1, 5:1, or 10:1 (parasite to host cells) for various times. ATP (5 mM; Invivogen) and lethal toxin, composed of 1 g/ml each lethal factor (LF) and protective antigen (List Biological Laboratories), were used as controls. …

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