Bacterial Toxin Research Citations

… Flat bottom microtiter plates (Nunc F96 Maxisorp) were coated with 50 l of Bacillus anthracis Protective Antigen (PA) or Lethal Factor (LF)(List Biological Laboratories (Campbell, Calif.) at a concentration of 1 g/mL in PBS overnight at 4 C. …

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 and Methods:

… Recombinant anthrax LF was obtained from List Biological Laboratories (Campbell, California, United States). PA was generously provided by Dr. Steven Leppla (National Institutes of Health). PA and LF were reconstituted in water at 500 ng/ml and 250 ng/ml, respectively [35]. Recombinant LF and PA were produced in B. anthracis and were free of LPS contamination as indicated by the manufacturer, and by a lack of CD86 up-regulation on immature cells, as measured by flow cytometry.

Cell death and viability assays:

Cell viability was measured by analysis of MTT cleavage to formazan by succinate dehydrogenases in living cells [37]. For the colorimetric MTT assay, cells were exposed to LT (500 ng/ml PA and 250 ng/ml LF), and the MTT solution (5 mg/ml MTT in PBS) was added directly to wells and incubated at 37 C for 4 h. The dye was solubilized with acidic isopropanol (25 mM HCl and 0.5% SDS in isopropanol), and the absorbance was measured at 570 nm. …

Western blotting:

Cells were cultured in 24-well plates and treated with LT.  …

In vivo assay:

Ten-week-old female BALB/c mice were injected intraperitoneally with LT [3,4]. Mice were sacrificed 0, 3, 6, and 24 h after LT injection. Spleens were harvested and treated with 400 U/ml collagenase D (Roche) to release DCs. Levels of splenic DCs (CD11c⁺, MHC class II⁺), macrophages (CD11b⁺, MHC class II⁺), and circulating B cells (B220⁺) and T cells (CD3⁺) were determined by flow cytometry using fluorescently labeled antibodies to surface markers.

LT (Lethal Toxin) = Lethal Factor (LF) + Protective Antigen (PA)

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:

Recombinant lethal factor (Prod #172), protective antigen (Prod #171), MAPKKide^TM (Prod #530), and MAPKKide^TM Unquenched Calibration Peptide (Prod #539) are products of List Biological Laboratories.

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor
• Product #539 – Unquenched Calibration Peptide for MAPKKide 530 for Anthrax Lethal Factor

Methods – The above materials were used in the following (refer to poster for details):

• HPLC
• Enzymatic activity
• Continuous fluorimetric assays
• LF-PA₆₃ binding assay
• LF enzymatic activity as a function of ZnCl₂

Materials and methods:

Rat LeTx challenge experiments were performed in accordance with protocols approved by the Scripps Institutional Animal Care and Use Committee. Male Fisher 344 rats (180200g; Harlan) were anesthetized with isofluoranes and were inoculated with LeTx mixture through a jugular-vein cannula. LeTx was prepared with 40 mg of PA and 8 mg of LF per rat (List Biological Laboratories). For rats that received sCMG2 and LeTx, sCMG2 was added to the LeTx mixture and was coinjected in a 500-mL volume. Rats recovered from anesthesia within 5 min and were monitored for symptoms of intoxication and death. Statistical analysis was conducted on the basis of Students unpaired t test (Prism, version 4; GraphPad). For rats that died overnight, statistical analysis was based on the last observed postinoculation time point.

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 AND METHODS:

… Recombinant LF and MAPKKide were both purchased from List Biological Laboratories (Campbell, CA). …

Fluorescence Peptide Cleavage Assay:

Cleavage reactions (100 l each) were performed in a 96-well plate. Each reaction contained MAPKKide (4 M) and LF (50 nM) in 20 mM Hepes, pH 7.4, and the small-molecule inhibitor. Kinetics of the peptide cleavage was examined for 30 min by using a fluorescent plate reader at excitation and emission wavelength at 485 and 590 nm, respectively.

The K_m and V_max values of the MAPKKide cleavage by LF were determined at 25C by using the same experimental condition described above for the fluorescence screening assay but with increasing MAPKKide concentrations (2, 3, 5, 8, and 10 M). The K_i and K_m(app) were calculated at a fixed 10 M inhibitor concentration. All constant values were definitely evaluated by fitting the data to the LineweaverBurk plot.

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 #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor

• Product #531 – MAPKKide Peptide Substrate (DABCYL/FITC) for Anthrax Lethal Factor

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