Citations

Reagents:

… BoNT/B light chain were purchased from List Biological Laboratories (Campbell, CA, USA). …

Assay for BoNT Endopeptidase Activity:

The cleavage of the recombinant GST-Synaptobrevin-II protein substrate by the BoNT/B light chain domain was performed as suggested by the manufacturer (List Biological Laboratories Inc., Campbell, CA, USA) with minor modifications. Briefly, 10 M of GST-Synaptobrevin-II protein substrate was incubated with or without 60 nM of BoNT/B light chain and were then were treated with 2.5 g individual mAbs or a combination of antibodies for 3 h at 37 C (control samples were not pre-treated with mAbs) in a 10 L reaction (20 mM HEPES, pH 7.4, 1.25 mM DTT, 0.3 mM ZnCl₂ and 0.2% Tween-20). …

• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant

Cell culture and reagents:

… Protective antigen (PA), lethal factor (LF) and Escherichia coli derived LPS were purchased from List Biological Laboratories Inc. (California USA). The following is a list of antibodies (Abs) used in this study. …

Long-term TIR cells were generated as previously described [110].  Briefly, RAW 264.7 macrophages were treated with a cytolytic dose of LeTx (250 ng/mL LF and 500 ng/mL PA) for 5 h and surviving cells were plated in fresh culture medium.  After two weeks, surviving clones were individually picked and plated on a 96-well plate.  Each clone was tested for LeTx sensitivity and resistant clones were pooled and propagated. Short-term TIR cells were generated as previously described [110].  Briefly, RAW 264.7 macrophages were treated with a sub-lethal dose of LeTx (100 ng/mL LF and 100 ng/mL PA) for 5 h and then supplemented with fresh media overnight.  The next day surviving cells were pooled and plated onto new culture plates with fresh media.  BMDM-TIR cells were generated by Dr. Soon-Duck Ha as previously described [177].  Briefly, BMDMs were treated with a sub-lethal dose of LeTx (100 ng/mL LF and 100 ng/mL PA) for 24 h and surviving cells were plated onto new culture dishes with fresh media. …

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

O s inibidor es Y – 27632 e Toxina A de Clostridium difficile fo ram obtido s da Calbiochem (San Diego, CA) e List Biological Labs (Campbell, CA, USA), respectivamente . …

• Product #152C – Toxin A from Clostridium difficile

Materials:

Standards of pure BoNT (150 kDa) were obtained from List Biological Laboratories, USA (BoNT/A) …

Experimental Procedures:

… To obtain high rates of successful dissection only the left phrenic nerve hemidiaphragms were excised from male mice of strain RjHan:NMRI … In each experiment, the preparation was first allowed to equilibrate for 15 min under control conditions. Then, the incubation solution was replaced by the toxin-containing solution (BoNT/A, B or E). After toxin application, the amplitude remained unchanged for some time (Figure 1), then decreased gradually depending on the toxin concentration. Toxin concentrations were such as to allow reduction of the contraction amplitude by 50% between 40 and 150 min. The times required to decrease the amplitude by 50% (paralysis time t_1/2 180 min) at different BoNT concentrations were used to construct the calibration curves for BoNT/A, B and E (Figure 2). Optimal power or logarithmic functions depending on the spread of the calibration curves were fitted to the calibration curves: y (BoNT/A; 0.031, 0.125, 0.5, 2, 8 pg/mL) = 46.387x^0.2622, …

Mice and immunizations

Eight‐ to ten‐week old C57BL/6 female mice were purchased from Japan SLC (Hamamatsu, Japan). For intranasal immunization of HBHA + CT, the mice were immunized with 30 μg of rHBHA and 1 μg of CT (List Biological Laboratories, Campbell, CA, USA) every 7 days, four times. Lymphocytes that had infiltrated the lungs were isolated and co‐cultured with APCs in the presence of HBHA. Two days later, cytokine concentrations in the culture supernatants were measured by ELISA. The cell cultures were performed in quadruplicate. For combined immunization, the mice were first s.c. immunized with 5 × 106 CFU of M. bovis BCG Tokyo strain (Japan BCG Laboratory) or PBS as a negative control. Twenty‐eight days later, the mice were intranasally immunized with 30 μg of rHBHA and 1 μg of CT or with PBS once a week four times….

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

-Galactosidase and CT assays:

… A positive-control assay for quantification of the level of CT in the samples was performed using purified CT (List Biological Laboratories).

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

Materials and Methods:

…CTB was visualized by incubating sections in a goat anti-CTB polyclonal antibody (List Biological Laboratories, 1 : 33 000 dilution) overnight, …

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

… in addition, there are several versions of FRET based peptides, for example, VAMPtide (O-Abz/Dnp), VAMPtide (FITC/DABCYL), … available on market (List Biological Laboratories, INC, USA)

• Product #540 – VAMPtide Peptide Substrate (o-Abz/Dnp) Substrate for C. botulinum Type B Neurotoxin
• Product #541 – VAMPtide Peptide Substrate (FITC/DABCYL) Substrate for C. botulinum Type B Neurotoxin

Induction of EAE and clinical evaluation:

An emulsion (0.2 ml) was prepared by mixing 0.1 ml of 200 µg PLP in phosphate-buffered saline (PBS) solution and 0.1 ml of complete Freunds adjuvant (CFA) containing killed mycobacterium tuberculosis strain H37RA (Difco, Detroit, MI) with a final concentration of 4 mg/ ml. On day 0, the emulsion was subcutaneously (s.c.) injected into SJL/J female mice (57 weeks old) at regions above the shoulder and the flanks (total of 4 sites; 50 µl at each injection site). Then, 200 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) was injected intraperitoneally (i.p.) on the day of immunization (day 0) and 48 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:

C57BL/6J mice were injected SC with 50 µg MOG p35-55 (Auspep, Parkville, Australia) emulsified in complete Freund’s adjuvant containing 200 µg heat-killed Mycobacterium tuberculosis on day 0. Mice received 200 ng Bordetella pertussis toxin (List Biological Laboratories, Campbell, CA) IV in 0.2 mL phosphate-buffered saline (PBS) on days 0 and 2. …

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)