Bacterial Toxin Research Citations

Materials and Methods:

… Recombinant protective antigen (rPA) of recent production from List Biological Laboratories Inc. was used as a control for these studies.

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

… Anthrax protective antigen-83 (PA83) was purchased from List Biological Laboratories. …

MPII Proteolysis of Proteins in Vitro:

Anthrax PA83 (2 g; 1 m) was co-incubated for 1 h at 37 C with increasing concentrations of MPII in 50 mm HEPES, pH 8.0, containing 1 mm CaCl₂, 0.5 mm MgCl₂, and 10 m ZnCl₂. The total volume of the reactions was 20 l. Where indicated, GM6001 (1 m) was added to the reactions to inhibit MPII. The cleavage reaction was stopped by adding a 5 SDS sample buffer. The digest samples were analyzed by SDS-PAGE using 420% polyacrylamide gel.

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

Chemicals and Reagents:

PA and LF were purchased from List Biological Laboratories…Batches of PA and LF had different potency, accounting for the different amounts used in different experiments, each of which included control data for the same toxin batch.

Toxin Treatment and Cell-Viability Assay:

Cells were seeded in a 96-well plate at a density of 2 10⁵ cells/mL 1 d before toxin treatment. Various concentrations of PA combined with a fixed concentration of LF (200 ng/mL) were added to the wells, and cells were incubated for 4 h at 37 C.

Immunofluorescence Microscopy.

PA protein was labeled with Alexa-Fluor 488 using the protein-labeling kit…

Biochemical Assay of PA Binding and Internalization.

Cells were exposed to 1 g/mL of PA at 4 C for 1 h for the binding assay and then were shifted to 37 C for the indicated time for the internalization kinetics 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

LT treatment of NRVM:

Approximately 24 h after NRVM plating, NRVM were treated with LT in a 2:1 ratio of PA:LF prepared in dispersion medium. PA (activated, 63 kD segment) and LF were purchased from List Biological Laboratories (Campbell, CA) and each reconstituted in glycerol at a concentration of 0.5 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

Human cytokine antibody array assay: 

After separation by centrifugation, WBCs were washed twice with complete RPMI 1640 medium, and the cells were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin. WBCs (5 10⁶ to 10 10⁶ cells per ml) were treated with ET (25 pmol PA plus 25 pmol EF), LT (25 pmol PA plus 25 pmol LF), …

The following materials were used in this study. B. anthracis EF, LF, and PA were purchased from 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).

• 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).

Enzyme-linked immunosorbent assay (ELISA):

A 50 L of a 5 g/mL solution of activated PA63 (List Biological Labs, Campbell, CA, USA) dissolved in 50 mM carbonate-bicarbonate buffer (pH 9.6) (Sigma-Aldrich) was used to coat each well of a 96-well transparent flat plate …

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

Reagents:

…Recombinant forms of Bacillus anthracis PA63, LF, and EF were obtained from List Biological Laboratories (Campbell, CA) and reconstituted per the manufacturer’s instructions. …

The following applications were performed using the above listed Anthrax products:

• Integration of recombinant PA₆₃ artificial membranes
• The pH dependence of PA₆₃ conductance and LF binding
• Effect of EF/ LF present during acidification
• Planar membrane rupture by recombinant proteins
• Planar membrane rupture by cationic polymers
• Planar membrane rupture by animal isolates
• Droplet membrane rupture by animal isolates
• Molecular modeling

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

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).

 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 Proteins and Other Reagents:

PA and EF were purchased from List Biological Laboratories (Campbell, CA).

• 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).

Materials and Methods:

Recombinant, nicked PA₆₃ as well as LF from B. anthracis were obtained from List Biological Laboratories Inc. (Campbell, CA, U.S.A). One mg of lyophilized PA₆₃ protein was dissolved …

Cell Culture, Cytoxicity Assay and Measurement of TNF-release:

…Cells were trypsinized and reseeded for at most 15-20 times. For cytotoxicity experiments, cells were seeded in 96-well-plates and incubated with PA₆₃+ LF together with the respective heterocyclic azolopyridinium salt in serum-free medium. After the indicated incubation periods, the morphology of J774A.1 cells was visualized by using a Zeiss Axiovert 40CFl microscope (Oberkochen, Germany) with a Jenoptik progress C10 CCD camera (Jena, Germany). The cytotoxic effect caused by PA₆₃/LF on this cell line was analyzed in terms of cell lysis. …

Membrane Translocation Assay:

The pH-dependent membrane translocation of LF through PA₆₃-channels was investigated on the surface of intact J774.A1 cells as originally described earlier (1, 2). Cells were grown in 96-well-plates and incubated for 30 min at 37C in serum-free medium with bafilomycin A1 (100 nM). During this period, some cells were treated also with 100 M of either chloroquine-based heterocyclic azolopyridinium salts (HA substances). Subsequently, the cells were incubated for 30 min at 4C with PA₆₃ (1 g/mL) plus LF (1 g/mL) to enable binding of these proteins to the cell surface. Thereafter, the medium was removed and cells were incubated for 5 min at 37C with acidic medium (pH 5.0, for control pH 7.5) containing the respective HA substance (100 M). The acidic medium was removed and cells were incubated at 37C in neutral medium containing bafilomycin A1 (100 nM) to prevent normal uptake of LF. Pictures were taken after 2 h and the toxin-induced cell lysis was analyzed exactly as described before.

Lipid Bilayer Experiments:

Black lipid bilayer experiments were performed as described previously [63] using a 1% solution of diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster AL, U.S.A.) in n-decane as membrane forming lipid. The instrumentation consisted of a Teflon chamber with two aqueous compartments separated by a thin wall. The small circular whole between the two compartments had a surface area of about 0.4 mm². The aqueous salt solutions were buffered with 10 mM MES, pH 6. All salts were obtained from Merck (Darmstadt, Germany). PA₆₃ was added from concentrated stock solutions after the membrane had turned black, to the aqueous phase to one side (the cis-side) of the membrane. The PA-induced membrane conductance was measured after application of a …

Titration Experiments

The binding of heterocyclic azolopyridinium salts to PA₆₃-channels was studied by titration experiments similar to those used previously to investigate binding of carbohydrates to the LamB-channel of Escherichia coli or binding of chloroquine or EF and LF, respectively, to C2II- and PA₆₃-channels in single- or multi-channel experiments [27], [31], [33], [42], [64]. PA₆₃-channels were reconstituted into lipid bilayer …

In most cases the different HA-substances caused full blockage the PA₆₃-channels. In case of only partial blockage of the PA₆₃-channels caused by a certain fraction of channels that did not respond to binding …

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

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).

Materials:

Recombinant PA, EF, and LF toxins were obtained from the List Biological Laboratories, Inc. … Stable toxin solutions of PA, EF, and LF were prepared according to the supplier’s technical note (List Biological Laboratories, Inc., 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).

• 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).