Bacterial Toxin Research Citations

3. Experimental

3.1. Assays

3.1.1. General
All biochemical analyses and cell assays were performed as previously reported by Lin et al. [22] and Turner, Nielsen et al. [14] with the following modifications.

3.1.2. SNAPtide Enzyme Activity Assay
The assay was conducted as previously described [22]. The final assay concentrations are as follows: SNAPtide substrate #523 = 4 µM; BoNT/A LC = 10 nM; assay buffer = 40 mM HEPES pH 7.4, 0.01% Triton X-100, 1% DMSO.

3.1.3. Screening
Initial compound screening was performed at room temperature with SNAPtide substrate #521 (List Labs, Campbell, CA, USA) at a compound concentration of 40 µM.

3.1.4. Endpoint Assay
In total, 500 nM BoNT/A LC was incubated with 5 µM of each compound for 30 min at 37 °C. At the indicated timepoints, 2 µL aliquots were quenched in 48 µL of SNAPtide #523 substrate (25-fold dilution) to give a SNAPtide assay final concentration of 20 nM BoNT/A LC and 0.2 µM compound.

• Product #521 – SNAPtide® Peptide Substrate (FITC/DABCYL) for C. botulinum Type A Neurotoxin
• Product #523 – SNAPtide® Peptide Substrate flP6(DABCYL/5-IAF) for C. botulinum Type A Neurotoxin

Tregs depletion and agent administration

To deplete Tregs, DEREG mice were injected intraperitoneally with 1 μg DT (List Biological Laboratories, inc, USA) 24 h before LPC injection, then 0.5 μg DT every third day until 10 days after LPC injection. Control mice were treated with PBS injection. Flow cytometry in spleens and blood were used to confirm Tregs depletion. …

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

… Diphtheria toxin, Unnicked, from Corynebacterium diphtheriae List Biological Laboratories Cat# 150 …

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

… Biotinylated recombinant PA83 (List Biological Labs, Campbell, CA) was bound …

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

… Briefly, MOG/CFA emulsion was subcutaneously injected into the axilla of mouse (10 mg kg-1), followed by i.p. injection of pertussis toxin (… List Biological Laboratories, Inc., Campbell, CA) on the day of immunization and 48 h post immunization. Body weight and EAE scores of mice were monitored every day. EAE scoring criteria were as follows …

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)

… SF were stimulated with human recombinant TNF … lipopolysaccaride (LPS) from Escherichia coli J5 (100 ng/ml; List Biological Laboratories), …

• Product #301 – LPS from Escherichia coli J5 (Rc)

… Where indicated, Foxp3+ Tregs were ablated in host mice via ip administration of diphtheria toxin (List Biologicals) at 50 mg/kg on the day of adoptive transfer …

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

Mice, peanut sensitization, and EFB-2 treatment

Six-week-old female C3H/HeJ mice purchased from the Jackson Laboratory (Bar Harbor, ME) were maintained in pathogen-free facilities at the Mount Sinai vivarium according to standard guidelines (57). Mice were intragastrically (i.g.) sensitized with 10 mg of homogenized peanut (PN) in 0.5 mL PBS containing 75 mg sodium bicarbonate, 10 µg of the mucosal adjuvant cholera toxin (CT) (List Laboratories, Campbell, CA), and 16.5 µL (1.1 µL/g body weight) of 80 proof Stolichnaya Vodka® (a source of food grade ethanol) to neutralize stomach pH and increase gastrointestinal permeability, three times during week 0 (50). Thereafter, sensitization was done weekly as above except that the CT dose given was 20 µg.

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

… Pertussis toxin (PTX) List Labs Cat# 180 …

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

Antigens for in vitro stimulation

Pertussis toxin (PT) and filamentous hemagglutinin (FHA), the two Bp antigens present in all aP vaccines (36), were selected for in vitro stimulation of blood cells in the Bp-specific whole blood intracellular staining (BpWB-ICS) assay to expand vaccine-induced Bp-specific precursor T lymphocytes. A Bp lysate (BPL) was also used to detect cellular immune responses to Bp antigens that are not included in aP vaccines. A genetically detoxified PT mutant, with an inactivated S1 subunit (R9K, E129A), was chosen for the in vitro T cell assay (LIST Biological laboratories Inc., Campbell, CA, USA, #184) to avoid the toxic activity on target cells (37). …

• Product #184 – Pertussis Toxin Mutant