Bacterial Toxin Research Citations

Materials and Methods:

Animals – All experiments were performed according to international guidelines and approved by the local experimental animal committee of the University Medical Center Utrecht (DEC-ABC, Utrecht). On postnatal day 9 (P9), C57Bl/6 mice pups were anaesthetized with isoflurane (4% induction, 2% maintenance), the right common carotid artery was occluded by thermo-cauterization, xylocaine (100 mg/ml; AstraZeneca, Zoetermeer, The Netherlands) was applied and the incision was closed. After a minimal 1 h recovery, the pups were exposed to 10% O₂ in N₂ for 45 min and returned to their dams. Sham-control animals underwent anesthesia and incision only. LPS+HI or LPS+sham control animals received an intraperitoneal (i.p.) injection of LPS (List Biological Laboratories, Campbell, Calif., USA) at a dose of 0.5 mg/kg 14 h before surgery. …

Author has specified the following LPS product used:

• Product #423 – ULTRA PURE LPS from Escherichia coli O55:B5 (Lot#4231A1)

Immunohistochemistry:

Tissue sections were incubated for 30 min at 37C in PBS, followed by 30 min in 0.2 m HCl and 1 mg/ml pepsin (Fisher Scientific), then permeabilized and blocked as described above. Sections were incubated for 3 nights at 4C in a mixture of primary antibodies against Cholera toxin subunit B (List Biological Catalog #703, RRID:AB_10013220; 1:5000) and…

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Antibodies and ELISA assays:

Heavy-chain specific goat anti-human IgA-HRP was purchased from InvivoGen (San Diego, California, USA). ELISA assays for detection of anti-tetanus toxoid or anti-EEHV1A gL antibodies were carried out as described previously [13]. Briefly, ninety-six well polystyrene plates (Immulon 4 HBX, Thermo Electron Corp., Milford, MA) were coated with either 1 ug of purified tetanus toxoid (List Biological Laboratories,US) at a 20 ug/mL concentration…

• Product #191 – Tetanus Toxoid from Clostridium tetani

… goat anti-CTB antibody (List Biological Laboratories, Campbell, CA, USA), …were used as primary antibodies. …

• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Cell Preparation, Reagents, and Stimulation:

In some experiments, cells were pretreated with Ac-YVAD-cmk (50 M, ALX-260028-M001, Enzo Life Sciences) for 30 min before S. Typhimurium infection or fliC transfection. Otherwise, cells were treated with anthrax protective antigen (1 g/mL, List Biologicals)…

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

Immunofluorescence microscopy:

… To detect the localization of GM1 gangliosides, TG cell monolayers were incubated for 5min with biotin-cholera toxin B subunit (CTB) (10µg/ml, List Biological Laboratories, Campbell, CA), rinsed thrice in RPMI 1640 medium, and then incubated with L. monocytogenes for 30min at 37°C. …

• Product #112 – Cholera Toxin B Subunit Biotin Conjugate

… For the rPA, 7.5 g of rPA (LIST Biological Laboratories) was mixed with alum and either saline or 50 g of Poly I:C was added to the mix on the day of immunization. Antibody assays. To measure levels of antibodies to the rPA …

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

Competition binding assay:

Apparent dissociation constants (K_d values) for the binding of actoxumab and bezlotoxumab to various purified toxins were determined by competition ELISA, as described previously (14). Briefly, high-protein-binding ELISA plates (Fisher Scientific, Waltham, MA) were coated overnight at 4C with 0.5 g/ml TcdA or TcdB (List Biological Laboratories, Campbell, CA), washed, and subsequently blocked for 1 h at 37C.

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

Toxoid vaccine preparation:

Vaccine for immunization of animals was prepared in the following way: lyophilized C. difficile toxins A (Product #152C) and B (Product #155) (List Biological Laboratories, Campbell, CA, USA) were separately reconstituted and then dialyzed into 20mM MOPS-HCl pH 7.4, 150mM NaCl, at concentrations of ~0.6mg toxin per ml.

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

…mixed with 10 µg of cholera toxin (List Biological Laboratories, Inc., Campbell, CA, USA) once a week for three consecutive weeks, or …

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