Bacterial Toxin Research Citations

EAU induction and clinical evaluation

Mice were immunized subcutaneously on day 0 with 300 µg of Interphotoreceptor Retinoid-Binding Protein (IRBP) 651–670 (Hanheng Group, Shanghai, China) emulsified in CFA (incomplete Freund’s adjuvant plus 4 mg/ml of Mycobacterium tuberculosis strain 37RA, Difco). Pertussis toxin (0.5 µg in 200 µl saline; List Biological Laboratories, USA) was administered twice intraperitoneally on day 0 and day 1 after immunization. Disease status was assessed by fundus examination on a scale of 0–4 based on the number, type, and size of lesions and extent of inflammation, as described previously [26].

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)

4.1. Animal Experiments

We purchased six-week-old female C57BL/6 mice from CLEA Japan, Inc. (Tokyo, Japan). …

We divided mice into two groups: the naïve and CFA-injected groups (eight mice per group). For the CFA-injected group, we sensitized mice subcutaneously with CFA that consisted of IFA (BD, Franklin Lakes, NJ, USA) and M. tuberculosis H37 Ra (BD) on days 0 and 19. The final concentration of M. tuberculosis in the CFA emulsions was 2 mg/mL (400 μg/mouse). We also injected CFA-injected mice with 300 ng of PT (List Biological Laboratories, Campbell, CA, USA) intraperitoneally on days 0 and 2. PT has been used by other research groups, including Johanson II et al. [25], for CFA-induced autoimmune models [64]. …

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)

Experimental autoimmune encephalomyelitis

EAE was induced in both male and female mice between 8 and 12 weeks of age. MOG35-55 peptide (100 μg, CSBio; CS0681) was emulsified in complete Freund’s adjuvant containing Mycobacterium tuberculosis (1 mg/mL, Sigma; F5881; M. tuberculosis (BD 231141) added to final concentration of 4 mg/mL) and was injected subcutaneously (100 μL volume) at the base of the tail. Pertussis toxin (200 ng, List Biologicals; 180) was administered i.p. on the day of and 1 day after MOG immunization. …

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

EAE induction and clinical scoring

EAE/optic neuritis was induced with MOG (MOG33-55, GenScript, cat. no.163913-87-9) peptide and PTX (List Biological Laboratories, Campbell, CA, USA, cat. no. 181) in 9-week-old female mice with a procedure modified from the established protocol.29,30,44 Briefly, 225 μg MOG33-55 peptide emulsified with incomplete Freund’s adjuvant (IFA; BD Difco, 263910) and 2.5 mg/mL mycobacterium tuberculosis (BD Difco, 231141) were injected subcutaneously for immunization twice at days 0 and 7, and 500 ng PTX in PBS (2.5 μg/mL) was intraperitoneally injected twice at days 0 and 2 to break down the blood-brain barrier. Mice injected with the same volume of Freund’s adjuvant emulsion without MOG33-55 were used as sham controls. …

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

Generation of hTEC cultures

Cell cultures of primary human TECs (hTECs) were established following the procedure originally described by Green et al33 with modifications. … To enrich for TECs, epithelial cell adhesion molecule (EpCAM)-positive cells were enriched by using anti-EpCAM (CD326) (clone HEA-125, Miltenyi, Bergisch Gladbach, Germany) staining and anti-APC microbeads (Miltenyi) separation. The TEC-enriched cell fraction was plated (2.5 × 104 cells/cm2) on a feeder layer of lethally irradiated 3T3-J2 murine fibroblasts (Kerafast, Boston, Mass) and cultured in a humidified atmosphere of 5% CO2 in growth medium composed of a mixture of Dulbecco modified Eagle medium and Ham’s F-12 (a 3:1 mixture), 10% FCS (ThermoFisher Scientific), insulin (5 μg/mL, Eli Lilly, Indianapolis, Ind), adenine (0.18 mM, Sigma, St Louis, Mo), hydrocortisone (0.4 μg/mL, Sigma), cholera toxin (0.1 nM, List Labs, Campbell, Calif), …

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

Comparison with previously reported detection methods for tcdA and confirmation by ELISA for TcdA

Two pairs of classic PCR primers for gene tcdA (Kato et al. 1999; Lemee et al. 2004) were compared with our method using 33 C. difficile RT017 isolates and 30 other types of clinical isolates preserved in our laboratory (Table S3). For inconsistent results, ELISA was employed for clarification. Briefly, negative control (ATCC43593), positive control (ATCC-BBA1803), and tested C. difficile (N20) isolates (Table S3) were anaerobically cultured in BHI liquid medium at 37 °C for 24 h. After centrifugation, the supernatant was taken and filtered through a 0.45 μm filter, then incubated overnight at 4 °C in a 96-well plate. The plate was washed with PBST(Phosphate Buffered Saline + Tween) and blocked with 5% bovine serum albumin at 37 °C for 2 h. Polyclonal antibody (List Biological Laboratories, Toxin A IgY, 1:1000 dilution) was used for the first hybridization, and horseradish peroxidase (HRP-conjugated goat anti-chicken IgY (Invitrogen, 1:5000 dilution) served as the secondary antibody. …

Induction and assessment of EAE

8-week-old male C57BL/6 mice were injected subcutaneously in the dorsal flanks with 3 mg/ml MOG35-55 peptide (300 μg per mouse) in CFA containing 5 mg/ml heat-killed Mycobacterium tuberculosis (500 μg per mouse) on day 0. Mice were injected with Pertussis toxin (150 ng per mouse, list labs) on days 0 and day 2 through the retroorbital vein. In Tivantinib prevention trails, Tivantinib was intraperitoneally injected into mice (10 mg/kg) beginning at day 0 to day 22 every two days. …

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)

Determination of intracellular cytokine induction following SIV-Gag peptide stimulation

… Staphylococcal enterotoxin A & B (SEB/A-List Biologicals stimulation at 250 ng ml−1 served as a positive control. Peptide diluent (1% dimethyl sulfoxide) served as the negative control. …

… Protective antigen List Biological Labs #171E …

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

Enzyme-linked immunosorbent assay

Nunc-immuno microtitre plate wells with Maxisorp surface (Thermo Fisher) were coated in triplicates with 30ng/well of full-length TcdA (List Biological Laboratories, Inc., Campbell, CA) in 0.05M carbonate–bicarbonate buffer pH 9.6, and incubated overnight at 4°C. Wells were blocked with 1% (w/v) bovine serum albumin in PBS for 1h at room temperature, washed with PBS/0.05% (v/v) Tween 20 and then incubated for 1h with decreasing concentrations of His6-tagged VHH constructs in PBS containing 0.2% bovine serum albumin and 0.05% Tween 20 (PBS-TB). …

• Product #152C – Toxin A from Clostridium difficile