Bacterial Toxin Research Citations

Analysis of TMV-PA Antigens for Reactivity against PA Specific Antibodies:

SDS-PAGE, ELISA and Western Blots – 
 
… For westerns, gels were transferred onto 0.2 um nitrocellulose
membranes (7 cm x 8.5 cm) in Tris-Glycine buffer (25 mM Tris, 192 mM Glycine, 20%
Methanol, pH 8.3) for 1 hour at 100V. Membranes were blocked with 2.5% non-fat milk
in TBS (20 mM Tris, 150 mM NaCl, pH 7.6) for 1 hour at room temperature. Primary
antibody (Goat anti-PA polyclonal, List Biologics Lot # 7712A2 …

… (c) Wash sections with 0.01 M PB three times for 5 min each time. (d) Bathe sections in primary anti-CTB antibody, for example, List (List Biological Lab) goat anti-CTB (1:5005,000) in 0.01 M PB containing 1 % BSA and 1 % TX-100 either overnight at 4 C or 35 h at RT. …

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

Induction of Active EAE in Mice by Immunization with MOG and Adjuvant:

EAE was induced in female wild-type C57BL/6 mice or MT and IL-10deficient mice on C57BL/6 background…All mice were given 400 ng of pertussis toxin (List Biological) intraperitoneally at 0- and 48-h postimmunization.

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 EAE:

68-week-old female C57BL/6 mice were immunized subcutaneously on day 0 with 75 µg/mouse of MOG3555 peptide emulsified in complete Freund’s adjuvant supplemented with 400 mg/ml Mycobacterium tuberculosis H37Ra (Difco). Each mouse was subsequently administered 250 ng pertussis toxin (List Biological Laboratories) intraperitoneal on days 0 and 2 after MOG₃₅₅₅ peptide 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)

Synthesis of cholera toxin-based vaccine

Cholera toxin (CT) was obtained from List Biologicals Laboratories (USA) …

Immunizations and injections of antibodies

C57BL/6 mice synchronized by Depoprovera® treatment were immunized under anesthesia by vaginal application of CTBOVA (4μg) + CT (1μg) in PBS at days 0, 14, and 21. NKp46+ ILC cells were depleted after intraperitoneal (i.p) injection of 200μL of anti-NK1.1 mAb (PK136 clone, 1/20 dilution of ascite fluid) at days -9 and 2 before each immunization. Such protocol insured at least a 98% depletion of NKp46+CD3- ILC cells in spleen, genital lymph nodes and vagina without affecting invariant natural killer T (iNKT) cells. NKG2D/NKG2D ligands blockade was performed by i.p injection of 500μg of mAb anti-NKG2D (CX5) at days -1 and +1 before and after each immunization. For dendritic cells tracking experiments, mice were immunized once with CTBA488 (40μg) and CT (1μg) and were sacrificed after 7 days.

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

EAE immunization:

The mice also received two 400 ng of intraperitoneal injections of pertussis toxin (List Biological Laboratories) in 100 µl of PBS immediately and 48 h later.

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)

Antibody variable region cloning and humanization:

The m1B7 and m11E6 variable region genes (17) were amplified from hybridoma cells by RT-PCR using degenerate primers and cloned into pAK100 as described (39). Positive clones were identified by monoclonal phage ELISA using a PTx-coated plate (1 mg/ml in PBS; List Labs), followed by sequencing.

ELISA and binding assays:

For indirect PTx ELISAs, a high-binding 96-well ELISA plate (Costar) was coated with 1 µg/ml PTx. The plate was blocked with milk for 1 hr, followed by incubation with duplicate anti-PTx antibody dilutions from 50 g/ml for 1 hr at 25°C. …

…PTx was injected in duplicate at 30 µL/min or 100 µL/min (m1B7, ch1B7), with concentrations between 5200nM diluted in running buffer (PBS or HBS, pH 7.4, 0.05% Tween). …

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)

Surgical procedures:

To visualize regenerating axons, cholera toxin subunit B (CTB; List Biological Laboratories) was injected using a protocol modified slightly from that in the previous report…After the sciatic nerve between the thigh muscles was exposed, a small incision was made on the perineurium just proximal to the trifurcation site. Four microliters of unconjugated CTB solution (0.1% in PBS) were slowly injected using the Hamilton syringe through the perineural incision and animals were killed 5 d after the injection.

p>Tissue processing and immunohistochemistry:

For immunohistochemistry, tissue sections were treated with 10% normal serum and 0.3% Triton X-100 for 1 h, and then primary antibodies dissolved in the same blocking solution were applied at 4°C overnight. The primary antibodies were…and goat anti-CTB (1:10,000; List Biological Laboratories).

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

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