Bacterial Toxin Research Citations

Western blot analysis:

Cell lysates and culture supernatants were obtained from cultures at the transition phase of growth (4 h). Four-milliliter culture samples were centrifuged at 10,000 g for 10 min. Preparation of cell lysates for Western blot analysis of AtxA was performed according to the methods described by Hammerstrom et al. (24) … Primary antisera (anti-LF [R. J. Collier] and anti-EF [R. J. Collier]) or antibody (anti-PA [List Biological Laboratories, Inc., Campbell, CA]) was added to TBS-T and allowed to react with the membrane for 1 h at RT. …

Chemicals and Antibodies:

… We used LT from List Biological Laboratories. …

Results-

LT enhances ETEC H10407 adherence to HCT-8 cells:

We showed previously that ETEC strains possessing LT adhere more avidly to cultured intestinal epithelial cells and induce the production of higher levels of cAMP, as compared with ETEC strains lacking LT (Johnson et al., 2009). To begin to determine the mechanism governing LT-induced adherence, we further developed assays using both the prototypical human isolate ETEC H10407 (Evans et al., 1975) and HCT-8 cells, a cell line derived from a human ileocecal colorectal adenocarcinoma (Tompkins et al., 1974) frequently used to study both ETEC … We also performed bacterial adherence assays to verify that LT expression enhances ETEC adherence to HCT-8 cells. Relative to the adherence of eltA ETEC (set to 1.0), wt ETEC adherence was increased 6.7 0.5-fold (Fig. 1B). This difference in adherence between wt and eltA ETEC was independent of the multiplicity of infection (moi) used in infection experiments (not shown). By exposing HCT-8 cells to purified LT holotoxin, we augmented eltA adherence to levels not significantly different (6.0 0.5-fold) from wt ETEC. By contrast, adding purified LT to wt ETEC induced no further increase in adherence. Thus, LT enhances both cAMP production and ETEC adherence to HCT-8 cells.

Lethal Toxin (LT) = LF + PA

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

The PA standard used in these experiments was obtained from List Biological Labs, Inc. and used at 1000 ng/mL concentrations.

Place Exchange of PA onto AuNPs:

For a ligand to peptide feed ratio (L/P) of 25:1 GS/PA, 20 mg GS AuNP was
added to 1.63 mg loop PA or 1.54 mg linear PA in 6.66 mL deionized water and stirred
for 3 days at room temperature.  For 25:1 TioEG/PA, 25 mg TioEG MPC was added to
3.94 mg loop PA or 3.72 mg linear PA in 25 mL deionized H2O at room temperature.

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

Antigens:

… Recombinant rPA from Bacillus anthracis were purchased from List Biological Laboratories, Inc. …

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

Analysis of cytokine and chemokine expression in BMDCs or in nasal septal tissues:

BMDCs were cultured for 5 days as described above. A total of 4 10⁶ BMDCs/treatment were stimulated with 0.001, 0.01, or 0.1% NE, 1, 10, or 30 g/ml CT (List Laboratories). As a positive control BMDCs were treated also with 1 or 10 ng/ml LPS Salmonella minnesota (List Laboratories) or left untreated as negative control. …

• Product #304 – LPS from Salmonella minnesota R595 (Re)

Author did not specify which Cholera toxin was utilized.  List Labs provides Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae and Product #101 – Cholera Toxin from Vibrio cholerae.

Materials:

Recombinant LF and PA were from List Biological Laboratories. …

SDS-PAGE-Based Cleavage Assay:

LF (25 nM in assay buffer) was mixed with compound at the indicated concentration, and the reaction was initiated by adding unlabeled MKK6 to 1 M and stopped after 30 min at RT by adding 4 SDS-PAGE loading buffer. …

Cytotoxicity Assay:

RAW264.7 cells were plated in 96-well dishes at 4  10⁵ cells per well and were allowed to recover for 16 hr, after which the medium was removed and replaced with fresh complete medium (100 l per well) containing 400 M compound or 4% DMSO vehicle control. After 30 min, PA (0.5 g/ml) and/or LF (0.0010.3 g/ml) were added and incubation continued for an additional 4 hr.

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

… These assays were conducted using 60.8 ng/ml of PA (List Biological Laboratories, Campbell, CA) and 48.6 ng/ml of LF (List Biological Laboratories) to achieve more than 99% cell lysis at a fixed cell density of 3 10 4 cells/well as determined by toxin potency testing. …

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:

… The Anthrax Lethal Factor and Anthrax Protective Antigen were obtained from List Biological Laboratories, INC.

Assay for the screening of the small molecule library against APA and ALF:

An analyzing solution consisting of 10 L of the Small molecule-carrying MRnS
and 2,000 L DI water was prepared. Samples containing different concentrations of free
toxin (APA or ALF, 2 pM to 20 nM, in 1X PBS buffer) were prepared and 2 L of each
sample was added to 200 L of the MRnS analyzing solution (Small molecule-MRnS). A
negative control sample was prepared in the same fashion, adding 2 L fresh 1X PBS
buffer instead of toxin (0 M APA or ALF control sample). Magnetic relaxation
measurements were performed every 15 minutes of incubation at room temperature for 1
hour. …

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

Synthesis of HD5 and Mutant Analogues:

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

LF Inhibition Kinetics:

The inhibition of LF by various defensins was quantified using an enzymatic kinetic assay (52). Briefly, freshly prepared LF at a final concentration of 1 g/ml (10 nm) was incubated at 37 C for 30 min with a 2-fold dilution series of defensin in 20 mm HEPES buffer containing 1 mm CaCl2 and 0.5% Nonidet P-40, pH 7.2. 20 l of LF substrate (1 mm) was added to each well to a final concentration of 100 m in a total volume of 200 l. The enzyme activity, characterized as a time-dependent absorbance increase at 405 nm due to the release of p-nitroaniline, was monitored at 37 C over a period of 5 min on a 96-well Vmax microplate reader …

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).

Phage display and phage ELISA:

… To measure the activity of phage-displayed scFvs, phage ELISAs were performed in high-binding 96-well plates (Costar) coated with 1 g/ml KJ126 monoclonal antibody (BD Pharmingen; to assess DO11.10 scFv activity), 16 g/ml anthrax protective antigen (List Labs; 14B7), …

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

Materials:

… Protective antigen was acquired from List Biologicals (Campbell, CA), …

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