Bacterial Toxin Research Citations

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. …

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

Materials and Methods

Anthrax lethal factor, Product #169 and MAPKKide® Plus, Prod #532 are from List Biological Laboratories,
Inc. The 96-well, black, flat bottom, nonbinding plates used for the fluorescent plate assay were from
Corning (cat # 3991). The filtered adult bovine plasmas: Na-Citrate, cat #7310806; K2-EDTA, cat #7310807;
K3-EDTA, cat #7310808; Li-Heparin, cat #7310810 ; Na-Heparin, cat #7310811; and the filtered adult bovine
serum cat #7330800 were purchased from Lampire Biological Laboratories.

Sample Preparation: Stock solutions of the fluorogenic substrate, MAPKKide® Plus, were made 1.25 mM
in DMSO. The substrate was then diluted 5-fold to 250 µM in assay buffer: 20 mM HEPES, pH 8.0 containing
0.1% Tween-20.

LF Activity Assay: LF was spiked into each plasma or serum sample and then each was diluted 1:5 using
assay buffer. The reaction was initiated by addition of 10 μM MAPKKide® Plus directly to the 1:5 diluted
plasma or serum, and the assays were run at 37°C using the kinetic mode of the plate reader with readings
at 1-minute intervals. The samples were run in triplicate with 9 replicate blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 triplicates).

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

… A total of 31.7 nM PA alone (List Labs, no. 171E) or in the presence of 13.6 nM LFN, LFN-ACD, or LFN-RID was added to the media, and cells were incubated for 20 hours at 37C in the presence of 5% CO2 …

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