Citations

Reagents:

Recombinant B. anthracis LF and PA (rPA) were acquired from List Biological Laboratories (Campbell, CA, USA). …

Immunization of rabbits and sheep:

Rabbits were immunized s.c. with rPA mixed with Freund’s complete adjuvant (FCA) initially, and with Freund’s incomplete adjuvant (FIA) at the time of boosting 3 and 6 weeks later. Rabbits were bled 1 week after the last immunization. …

Cytotoxicity neutralization test:

LeTx (LF=16 ng ml¹ or 64 ng ml¹; PA=500 ng ml¹) was pre-incubated in DMEM with varying concentrations of the tested antibody for 1 h at 37C in a 5% CO₂atmosphere. …

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:

… Anthrax protective antigen was purchased from List Biological Laboratory (Campbell, CA), …

Purification of EF, EF3, CyaA-N, and CaMEF3 and CyaA-N, the catalytic domains of EF and CyaA, respectively, as well as calmodulin were purified as described previously (37,38). To express edema factor that has a hexahistidine tag substituted for its leader peptide (amino acids 133) and can be delivered by anthrax-protective antigen into host cells, a plasmid, pProEx-H6-EF, was constructed as follows. The 3.2-kb EcoRI-XhoI fragment was excised from pSE42 (kindly provided by S. Leppla, National Institutes of Health) and inserted into pBluescript. A NotI site was then introduced at the sequence encoding amino acids 3234 of EF by site-directed mutagenesis, and the mutation was confirmed by DNA sequencing. 

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

PA-Binding Studies:

PA binding to CHO-R1.1 cells that expressed CMG2⁴⁸⁹-EGFP or ATR/TEM8 sv2-EGFP was assessed by flow-cytometric analysis; 10⁶ cells of each type were incubated with 50 nM PA (1 h, 4C), washed with PBS, incubated with a polyclonal PA-specific rabbit antiserum (9) (1:500 dilution, 1 h, 4C), washed with PBS again, and then incubated with an allophycocyanin-conjugated anti-rabbit antibody (Molecular Probes; 1:2,000 dilution, 1 h, 4C). ELISAs were performed by binding 0.3 g of PA83 (List Biological Laboratories, Campbell, CA)/100 l of TBS in a well of a MaxiSorp plate (Nalge). Samples were then treated with TBST solution (TBS containing 3% BSA and 0.05% Tween 20), washed with TBST, and incubated with 0300 ng of purified CMG2^VWA/I-MycHis/100 l TBST in the absence or presence of 1 mM MgCl₂, MnCl₂, CaCl₂, or ZnCl₂. The samples were then washed with TBST and incubated with a horseradish peroxidase-conjugated anti-His antibody (1:2,000 dilution; Santa Cruz Biotechnology). All incubations for ELISAs were performed for 1 h at room temperature. The levels of bound antibody were then measured by using Supersignal ELISA Pico chemiluminescent substrate (Pierce) and a luminometer (Victor V, Wallac).

Intoxication Assays:

Cell viability assays (WST-1 assay; Roche Molecular Biochemicals) were performed in triplicate by incubating 5,000 cells of each type for 30 h with 2 10¹⁰ M LF_N-diptheria toxin A chain (DTA) and varying concentrations of PA (10¹² to 10⁸ M; List Biological Laboratories) or no PA (100% viability control). The inhibition assays were performed as above with different amounts of CMG2^VWA/I-MycHis (01,000 ng/100 l) added to 10⁹ M PA and 10¹⁰ M LF_N-DTA before this mixture was added to cells. The IC₅₀ was determined by regression analysis (prism, GraphPad, San Diego).

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

Materials:

… The purified recombinant anthrax lethal factor (LF) and both MAPKKide substrates are products of List Biological Laboratories.

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