Bacterial Toxin Research Citations

…In groups 1, 2, 3 and 5, vaccine formulations consisted of Hank’s balanced salt solution (HBSS) with 5 µg cholera toxin (CT) (List Biological Laboratories Inc., Madison, NJ, USA), …

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

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).

Antigens and EAE Induction:

…Each mouse received 0.05 ml emulsion (s.c.) at four sites and pertussis toxin (300 ng i.p.; List Laboratories, Campbell, CA) at 0 h and 72 h post immunization. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

EAE induction and TFA-12 treatment:

Mice were given intravenous injections of pertussis toxin (List Biological Laboratories) on the day of the immunization and 48 h later and were divided in two experimental groups.

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Induction of experimental autoimmune encephalomyelitis (EAE):

EAE was induced in C57BL/6 wild type, Panx1^+/+ and Panx1^/ mice by subcutaneous immunization with 300g of myelin oligodendrocyte glycoprotein MOG₃₅₅₅ peptide (MEVGWYRSPFSRVVHLYRNGK;… The day of MOG immunization was designated day 0. On day 0 and day 2 post immunization (dpi), mice were injected intraperitoneally with 500ng Pertussis toxin (List Biological Laboratories, Campbell, CA, USA).

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Cholera toxin B subunit (CTB) tracer injection:

After 67 d, a CTB tracer was injected in the sciatic nerve. Anesthesia was induced in all rats with 5% isoflurane and maintained at 2.5%. The sciatic nerve of the left hindpaw was exposed, and each of the two branches was injected with 1 µl of CTB solution (#104; List Biological Laboratories; 10 mg/ml)…

Immunohistochemical labeling of CTB:

To examine the regeneration of the dorsal column fibers, animals were killed 10 weeks after the lesion and 3 d after CTB injection and CTB signal was visualized and quantified as follows…blocked with TBS containing 2% horse serum (Invitrogen) and 0.3% Triton X-100 (blocking buffer) for 45 min and incubated at 4C with goat-anti-CTB (#703, List Biological Laboratories; 1:80,000)

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Toxin, Antibodies, and Reagents:

… The anti-PA antibody was purchased from List Biological Laboratories, Inc (Campbell, CA, USA). …

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).

Production and purification of rPA and rLF preparations:

MLA:

The J774A.1 mouse macrophage cell line (ATCC; TIB-67, Manassas, VA) was cultured in 75-cm² flasks in Dulbecco’s modified Eagle’s medium with high glucose … Culture medium was discarded before incubation with rPA and rLF. The macrophage lysis assay (MLA) using different concentrations of rPA and rLF was performed by diluting reagents in appropriate volumes of complete DMEM.

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

… The Strep-tag of rCdtA was cleaved with enterokinase, followed by repurification of CdtA on Ni-NTA agarose to remove the cleaved tag and the enterokinase. Native toxins A and B were purchased from List Biological Laboratories. …

• Product #152C – Toxin A from Clostridium difficile
• Product #155 – Toxin B from Clostridium difficile