Bacterial Toxin Research Citations

Toxins:

PA+FP59 was the toxin for cytotoxicity assays. … PA was also purchased from List Biological Laboratories (Campbell, CA).

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

SNAPtide assay of BoNT/A LC with screened compounds:

SNAPtide (o-Abz-Dnp) was purchased from List Biological Laboratories and was used
for analyzing the kinetics of protease inhibition.  The BoNT/A LC is included in a mixture
(denoted Big Mix) to minimize discrepancies in concentration of enzyme and to have a constant
concentration of BoNT/A LC throughout the whole experiment.  Big Mix includes 1xPBS,
20M Zn2+, and 0.2M LC/A-SUMO.   Compounds B, D, and E were dissolved in DMSO and
the LC Buffer (1xPBS, 20M Zn2+) and DMSO was tested as a negative control to determine any
possible interference in fluorescence.  The catalytic activity of BoNT/A LC is largely insensitive
to the presence of DMSO, making this an ideal co-solvent (Boldt et al, 2006).   The SNAPtide
assay was initiated by the addition of 2M SNAPtide and the reaction was allowed to proceed to
5% completion and the initial rate was determined.  The protease activity was monitored by the
increase of donor fluorescence intensity.  The compound 7-(phenyl(8-quinolinylamino)-8
quinolinol, better known as QAQ (84096), has previously shown in an HPLC-based enzymatic
assay to inhibit BoNT/A LC by 74% at 20M enzyme concentration (Lai et al, 2009). …

• Product #520 – SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin

EAE Mice Model and Treatment Protocols:

C57BL/6 mice were obtained from the Animal Center of Third Military Medical University. All experiments were performed in accordance with Health Guide for the Care and Use of Laboratory Animals, with the approval of Third Military Medical University Committee on Animal Care (permission NO: SCXK-JUN-2007-015). Female C57BL/6 mice (N=43, 8 weeks old) were immunized subcutaneously with 200 µg of MOG₃₅₅₅ peptide (Invitrogen, Carlsbad, CA) emulsified in complete Freund adjuvant (CFA, Sigma Aldrich, Saint Louris, MO) on Day 0 and Day 7, and received 300 ng pertussis toxin (PT, List Biological Laboratories, Campbell, CA) in 0.1 ml PBS intraperitoneal at the time of immunization and 48 hours 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)

Immunization with Purified Antigens:

As previously described (38), mice were immunized intranasally with 4 µg of recombinant protein and 1 µg cholera toxin as adjuvant (List Biological Laboratories) per 20 µL dose. Control mice received adjuvant alone. Three immunizations were given at weekly intervals, followed by intranasal S. aureus challenge at week 5, as described above.

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

Adjuvants:

B. pertussis toxin and DNA containing unmethylated CpG-dinucleotide were purchased from List Biological and Operon, respectively. NE was made at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. This adjuvant is composed of a water phase (PBS, Tween 80) and oil phase (Squalene with or without -tocopherol) at a 4:1 ratio. The nanoemulsion was prepared by passing the mixture through M-110 PMicrofluidizer (Microfluidics) under a pressure of 18,000 psi. …

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)

Animal models and ethics statement:

… All immunizations were performed by instilling 20 µl of a mixture of PBS, 1 µg of cholera toxin (CT; List Biologicals, Campbell, CA) and either 4 µg of the indicated purified pneumococcal protein or 100 µg of WCC as immunogens onto the nares of gently restrained unanesthetized mice. Control animals were immunized with CT mixed in PBS alone to control for any nonspecific effect of the adjuvant alone. Animals were immunized intranasally twice at one-week intervals. …

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

… After this, cells were preincubated for 30 min with 50 ng/mL of IL-10, 5PEGIL-10 or 20PEGIL-10 in 0.1 mL FBS-free medium containing 0.5% normal mouse serum. At t = 0, 25 ng/mL of LPS (E. coli, List Biological Laboratories, INC., USA) was added to the wells. …

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

Materials:

The following TLR agonists were purchased: smooth LPS from E. coli O55:B5 (List Biologicals) …

Macrophage culture and cytokine assays:

BMDMs were washed three times in serum-free medium, followed by incubation overnight with HMGB1, with or without hemoglobin, or with different TLR agonists as desired at indicated concentrations.NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) was utilized.

NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) was utilized.

Product #203 (5 mg – LPS from Escherichia coli O55:B5) is no longer sold.

Product #203A (2.5 mg – LPS from Escherichia coli O55:B5) is available for purchase.

• Product #203A – LPS from Escherichia coli O55:B5

Cell culture:

Bone marrow-derived macrophages (BMDM) were prepared by harvesting marrow from femurs and tibias of 129+/+ and Nlrp1b2/2 mice. Marrow was cultured for 7 d in IMDM containing 15% L cell-conditioned medium, 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). BMDM were incubated in OPTIMEM plus Glutamax (Life Technologies) containing varying concentrations of LT (List Biological Laboratories, Campbell, CA) for 4 h, and cell viability was assessed with the WST-1 reagent (F. HoffmannLa Roche, Basel, Switzerland). Peritoneal macrophages were elicited from wild-type, Nlrp1b2/2, Nlrp32/2, and Casp-12/2 mice with an i.p. injection of 4 ml 4% thioglycollate in PBS. Seventy-two hours after injection, the peritoneal cavity was lavaged with DMEM, and cells were plated and incubated for 1 h. Adherent cells were then washed twice with cold PBS and cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). For LT treatments, macrophages were primed overnight with 100 ng/ml ultrapure LPS (Invitrogen Life Technologies), washed with PBS, and incubated with OPTIMEM plus Glutamax containing either PBS or 1 mg/ml LT for 6 h. …

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

Materials:

… Ultrapure LPS (TLR4 ligand) isolated from Salmonella minnesota R595 was obtained from List Biological Labs (Campbell, CA). …

Cell culture and ELISA:

… Monocytes were isolated with .95% purity, based on CD14 positivity in ow cytometric analysis and were maintained in RPMI 1640 supplemented with 10% FCS. Cells were incubated with TLR ligands PAM2, PAM3, and LPS, or with M. tuberculosis whole cell lysates for 24 h. …

• Product #434 – ULTRA PURE LPS from Salmonella minnesota R595 (Re)