Bacterial Toxin Research Citations

… Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free) List Biological Cat#181 …

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)

EAE

EAE was induced as previously described52. In brief, 8–12-week-old female C57Bl/6J mice were immunized using 150 μg of MOG35–55 (Genemed Synthesis, 110582) mixed with freshly prepared Complete Freund’s Adjuvant (using 20 ml of Incomplete Freund’s Adjuvant (BD Biosciences, no. BD263910) mixed with 100 mg of Myobacterium tuberculosis H37Ra (BD Biosciences, no. 231141)) at a ratio of 1:1 (v/v at a concentration of 5 mg ml−1). All mice received two subcutaneous injections of 100 μl each of the MOG35-55/CFA mix. All mice then received a single intraperitoneal injection of 200 ng of pertussis toxin (List Biological Laboratories, no. 180) at a concentration in 200 μl of PBS. …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

Tetanus toxin labeling

Alexa Fluor®647 was attached to the tetanus toxin (List Biologicals cat. no. 19050A2) according to the manufacturer’s protocol from Alexa Fluor®647 Labeling Kit (Microscale Protein Labeling Kit, cat. no. A30009; ThermoFisher Scientific) and stored at −20°C. Quantification of the Alexa Fluor® 647 ratio per tetanus toxin was obtained by scanning measurements using the NanoDrop One c device (Thermo Fisher Scientific) with 280 and 650 nm readings. The average dye per mole of protein obtained was 1.7 moles.

Tetanus toxin internalization inhibition assay

The TeNT entry inhibition by mAbs was initiated by the pre-incubation of 25 nM TeNT-Alexa-Fluor 647 and 25 nM or 50 nM mAbs in Neurobasal-A medium on an orbital shaker at 37°C, 5% CO2 for 1 hour. The mixtures and increased concentrations of TeNT-Alexa-Fluor 647 (25 nM, 50 nM, and 100 nM) were added to cells for 1 hour at 37°C and 5% CO2. Cells were washed twice in 2% formaldehyde solution in PBS and processed for immunofluorescence.

• Product #190 – Tetanus Toxin from Clostridium tetani

Mucosal sensitisation model

The animal study protocol was approved by the “Service Vétérinaire du Canton de Vaud”, Switzerland and the experiments were performed at Ambiotis (Toulouse, France). The model has been previously described in detail (Zuercher et al., Citation2012). Briefly, five-week-old conventional female BALB/cOlaHsd mice from Envigo (Gannat, France) were sensitised for a total of seven consecutive weeks by weekly gavage of 50 mg ovalbumin (OVA, Merck) [sensitised group (S.)] or water [non-sensitised group (N.S.)] in combination with 20 μg cholera toxin (CT) (List Biologicals, purchased from LuBioscience, Lucerne, Switzerland). On day 67 or 68, mice were challenged with an oral administration of 200 mg ovalbumin (OVA; Sigma, Switzerland) and harvested at day 68 or 69 (Supplementary Figure 1A). Two independent experiments were performed with n = 8 animals per group for the exploratory experiment and n = 13 animals per group for the confirmatory experiment.

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

Drugs and Pharmacology

Formalin (2%, Sigma), PEG (30%, average M.W. 1500, Acros Organics), CFA (50%, Sigma), LiCl (12.5 mg/mL, Sigma), CNO (0.1 mg/mL, Tocris) and Diphtheria toxin (DT, 0.5mg/ml, List Labs) were diluted in normal saline. … DT was injected intramuscularly at a volume of 5 µL/g body weight. Mice received 3 DT injections under light isoflurane anesthesia with 48 h between injections.

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

… Goat anti-CTb (1:5000, IHC) List Biological Labs 703 …

CTb injections

Hindpaw: Mice were anesthetized (5% induction, 2-3% maintenance). Injections of 1% unconjugated CTb
(~7µL across four sites) were injected into the ventral paw of C57BL/6 wild type mice to label paw sensory
input. Animals were perfused 5 days after CTb injection and tissue was processed for IHC as described above.
Free floating sections were immunostained with goat-CTb (list-labs). 5x images of CTb-labeled afferent inputs
were taken at the Zeiss LSM 800 confocal microscope and ImageJ was used for synaptic analysis.

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

… Rorctd Tomato DTR mice were injected with diphtheria toxin (10 ng/g, i.p.; List Biological Laboratories, Campbell, CA, USA) using single or continuous injections, as indicated …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

… The fluorescence intensity was analysed using FlexStation 2 (Molecular Devices, Ex/Em = 490/525nm). Where appropriate, cells were treated with PTX (100ng/ml; List Biological Laboratories) for 16?h and with YM-254890 (1µM; a gift from Dr. J. Takasaki60) for 45min before the assay. …

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)

Toll-like receptor (TLR) 4 and 5 activity assays

TLR4 and TLR5 activity assays were performed as described previously with minor modifications (14, 15). Briefly, HEK-Blue-hTLR4 cells and HEK-Blue-hTLR5 cells carrying a secreted embryonic alkaline phosphatase (SEAP) reporter construct were obtained from InvivoGen (CA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin with and without 0.2% Normocin (InvivoGen, CA) for TLR5 cells and TLR4 cells at 37°C with 5% CO2, respectively. Monolayers of 1 × 105 cells per well in a 96-well plate were incubated with media alone, PBS, and FlaA and FlaB proteins at different concentrations ranging from 10 pg/mL to 10 µg/mL for 24 h. E. coli lipopolysaccharide O111:B4 (List Biological Laboratories, Inc., CA) was added to HEK-Blue-hTLR4 cell as a positive control. …

• Product #201 – LPS from Escherichia coli O111:B4

Tdap-specific IgG and IgG subclass detection

All available leftover maternal and cord serum samples from both deliveries were tested at the Université Libre de Bruxelles (ULB). Total and subclass Tdap-specific IgG antibodies were quantified using an in-house bead-based multiplex immunoassay (28). In brief, serum samples were incubated with distinct fluorescent magnetic beads, individually coated with specific purified target antigens: PT (List Labs, #180), FHA (Sigma, #F5551), PRN (NAC, #NAT41666), diphtheria toxin (DT, List Labs, #151) and tetanus toxin (TT, MassBiologics, # LP1105P). Samples were measured at appropriate dilutions with a Multigam reference (CAF-DCF, Belgium), a WHO international standard NIBSC 06/140 and blanks included on each plate. Serum dilutions used to titrate IgG and IgG1 were 1:9000-1:18000-1:36000, 1:70-1:140-1:280 for IgG2 and IgG3, and 1:70-1:700-1:7000 for IgG4. After washing, the captured IgG was detected with R-phycoerythrin labelled antibodies specific for each isotype (IgG1-4). …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #151 – Diphtheria Toxoid, from Corynebacterium diphtheriae