Bacterial Toxin Research Citations

Chemicals and reagents

… Toxins (product numbers) purchased from List Biological Laboratories were: EF (178A), diphtheria toxin (150), Clostridium septicum alpha toxin (116L), C. perfringens epsilon toxin (126A), C. difficile toxins A (152C), B (155B), and binary toxin subunit A(157A), Pasteurella toxin (156), pertussis toxin (181), botulinum neurotoxins A (612A) and B (622A) (heavy chains), and cholera toxin (100B). …

• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis
• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae
• Product #152C – Toxin A from Clostridium difficile
• Product #155 – Toxin B from Clostridium difficile
• Product #156 – Pasteurella multocida Toxin
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #612A – Botulinum Neurotoxin Type A Heavy Chain, Recombinant, Binding Domain
• Product #622A – Botulinum Neurotoxin Type B Heavy Chain, Recombinant, Binding Domain
• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Protective Antigen Binding Assays

For all PA binding assays, activated PA (63 kDa, List Biological Laboratories, Inc.; Campbell, CA, USA) was labeled using an Alexa Fluor® 647 Microscale Protein Labeling Kit (Molecular Probes; Waltham, MA, USA) following the manufacturer’s instructions. RAW 264.7, human BAL cells, and human leukocytes were processed as described for anthrax surface receptor expression through the point of Fc receptor blocking. At this point, cells were either left untreated, or incubated with 3000 or 10,000 ng/mL unlabeled PA as indicated for 30 min on ice. Cells were then exposed to AF647-labeled PA at 10, 100, or 1000 ng/mL for an additional 30 min on ice. In the case of lung and blood cells, antibodies used for cell subset-specific identification were also added at the same time as AF647-PA. All samples were centrifuged at 300× g for 5 min and washed twice with 1X PBS-2. Samples were fixed with 2% paraformaldehyde prior to flow cytometry.

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

Protective Antigen (PA) List Biological Laboratories, Inc., Campbell, USA

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

Toxin Neutralization Assay (TNA)
The functional ability of antibodies from vaccinated cattle and controls to neutralize anthrax lethal toxin was assessed using an in vitro TNA, as previously described by Ndumnego et al. [16]. The TNA was determined with J774A.1 mouse macrophage cells (ECACC cat no 91051511) exposed to anthrax lethal toxin in the presence of antibodies. Briefly, 1.0 × 105 J774A.1 macrophages per well in Dulbecco’s Modified Eagle Media (DMEM) and 10% foetal bovine serum (FBS) were incubated at 37 °C and 5% CO2 overnight in 96-well cell culture plates. Sera from each animal were 2-fold serially diluted in duplicates with the starting dilution of 1:50 in DMEM formulated with 5% FBS containing PA (500 ng/mL) and LF (400 ng/mL) (List Biological Laboratories Inc, Campbell, CA, USA) for 1 h at 37 °C and 5% CO2 before addition to the overnight-cultured confluent J774A. …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

Detection of anthrax-specific antibody levels

Anthrax-specific antibody levels against protective antigen were measured by ELISA12. High binding ELISA plates were coated with 100 ng per well of anthrax protective antigen (List Biological Laboratories Inc., Campbell, CA, USA) in carbonate buffer, pH 9.6 and incubated at 37 °C for 1 h, then overnight at 4 °C. …

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

… Anthrax lethal factor (#172C) and Anthrax protective antigen (#171E) were from List Biological Labs, Inc. …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

… Anthrax Protective Antigen List Biologicals Ref: 771B…

Chemicals, Peptides, and Recombinant Proteins

Protective antigen (PA), recombinant from Bacillus anthracis List Biological Laboratories 171C

Stimulation assays

… For the stimulation of the NLRC4 inflammasome, human macrophages were primed with 200 ng ml-1 LPS for 3 hours before 2 μg ml-1 LFn-PrgI and 0.5 μg ml-1 PA (from Bacillus anthracis) were added to the culture medium for 2 hours (Zhao et al., 2011).

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

Antibody quantification by ELISA

PA, LF and EF ELISAs were performed using commercially available antigens (List Biologicals, Campbell, CA) as previously described [21]. Cutaneous anthrax and regional control group sera were titered using 2-fold dilutions, beginning at 1:10. US control sera were titered at 10-fold dilutions, beginning at 1:10. …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis

… B. anthracis lethal factor (LF) was acquired from List Biologicals. …

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis