Bacterial Toxin Research Citations

… anthrax lethal factor (List Biological Labs #LL-169B) and protective antigen for anthrax lethal factor (LF) …

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

2.6. Toxin Neutralisation Titre (TNA) for Neutralising Antibodies Titre

The in vitro toxin neutralisation ability of vaccinated cattle antibodies was determined as previously described by Ndumnego et al. [38]. Briefly, the TNA was determined with 1.0 × 105 of J774A.1 mouse macrophage cells (ECACC cat no 91051511) per well followed by overnight incubation at 37 °C and 5% CO2. Then sera from vaccinated animals were 2-fold serially diluted in duplicates starting from the dilution of 1:50. Then the macrophages were exposed to anthrax lethal toxin (PA: 500 ng/mL and LF: 400 ng/mL (List Biological Laboratories Inc., Campbell, CA, USA) in the presence of antibodies. …

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

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

… Bacillus anthracis protective antigen (List Biological Laboratories, cat. no. 171E) …

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

… Test sera were duplicated and diluted two-fold starting with a 1:50 PA 500 ng/mL in lethal factor (LF) 400 ng/mL (List Biological Laboratories Inc., USA). …

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

… and active and inactive FlaTox (2 mg/ml) in combination with protective antigen (2 mg/ml) (List Biological Laboratories Inc., catalog no. 171E) for NLRC4 inflammasome activation …

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

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