Bacterial Toxin Research Citations

Antigen:

Recombinant PA, produced in an avirulent, non-capsulated, sporulation suppressed Ba host was obtained from List Biological Laboratories, Inc. (Campbell, CA). The PA migrated as a single major band with an apparent molecular weight of 83 000 daltons on 10% polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS). The PA was reconstituted in distilled water and stored aliquoted at 220C. Before use, individual aliquots were thawed and used immediately.

Competitive inhibition:

To determine the specificity of measurements performed by FCMIA, pre-incubation of positive sera, control sera, negative worker sera, and a dilution of AVR414 with PA (competitive inhibition) was performed. Sera (130 ml final volume) were treated with either 80 mg/ml PA (final concentration) or dilution buffer and incubated overnight at 4C. The sera were then centrifuged and the supernatants analysed as outlined above. …

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

ReagentsRecombinant anthrax PA and lethal factor LF were purchased from List Biological Laboratories, Inc. (Campbell, CA) and were stored as 1 mg/ml stock solutions in 1:1 glycerol:water. …

Cytokine induction by LT:

Figure 1:  Relative cytokine levels in the supernatants of RAW264.7 (A) and J774A.1 (B) cells are shown following 24-h treatment with 1 g/ml LF + 2.5 g/ml PA. Supernatants were harvested and tested for cytokine levels by ELISA in duplicate. The y axis values represent the ratio of induction of the various cytokines (gray bars) compared with unstimulated cells (black bars, arbitrarily assigned a value of 1). Shown is a one representative of two cytokine screening experiments. In separate experiments, extracellular levels of IL-1 and IL-18 were measured by ELISA in the supernatants of RAW264.7 (C) or J774A.1 (D) cells stimulated with or without LF (1 g/ml) and/or PA (2.5 g/ml) for 24 h. Bars indicate intraassay standard deviation.

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

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