Citations

Lethal Factor protease assay:

Lethal Factor (20 nM final concentration) and MAPKK substrate (MAPKKide 12.5 M, final concentration) were purchased from List Biological Laboratories, Campbell, CA and used according to the fluorescence resonance energy transfer (FRET) method. …

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

Sandwich ELISAs for LF detection:

Recombinant PA₈₃ and PA₆₃ were purchased from List Laboratories (New Jersey). PA₆₃ is a cleavage product that is capable of binding LF (45). For the sandwich ELISA, 50 l of PA at 63 kDa and 83 kDa (6 g/ml) was applied to a 96-well plate and blocked with 2% milk-PBS as described above. For initial assays, LF was diluted in PBS or human serum at 5 g/ml. For assays detecting LF in infected animals, serum was added to the plate initially diluted 1:1 in 2% milk-PBS and then serially diluted across the plate in duplicate. After a 1-h incubation, the plate was washed as described above. Goat anti-LF polyclonal serum (List Labs) was diluted 1:1,000 in 2% milk-PBS and added to the plate for a 1-h incubation in duplicate. The plate was then washed, followed by the addition of goat anti-rabbit IgG-HRP conjugate (Bio-Rad) diluted in 2% milk-PBS for 1 h. ELISA reactions were developed with OPD tablets (Sigma) and quenched by the addition of 50 l of 4.5 M H₂SO₄. …

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis
• Product #771B – Anti-Protective Antigen from B. anthracis (Goat)

Reagents and antibodies:

Recombinant anthrax PA and LF were purchased commercially and were stored in 1:1 glycerol-water at 20C (List Biological Laboratories) for in vitro studies. Unless otherwise indicated, anthrax LT was administered in excess at concentrations of 2.5 g/ml PA and 1 g/ml LF. In selected experiments a proteolytically inactive mutant of LF was used as a negative control (E687C substitution in zinc binding site that eliminates enzymatic activity; List Biological Laboratories). …

Anthrax PA binding assays:

Purified murine or human B cells were cultured at 4C for 30 min with FITC-labeled anthrax PA (50 g/ml; List Biological Laboratories) in the presence or absence of unlabeled anthrax PA (150 g/ml) to confirm specific binding. Stained cells were then washed with PBS and analyzed by flow cytometry (see below). Unstained cells were analyzed in parallel to establish background levels of autofluorescence.

ELISA:

Primary B cells were cultured in complete RPMI for 4 to 5 h, washed with RPMI 1640, and then stimulated as indicated in the presence or the absence of anthrax LT for 7 days.  …

Murine in vivo studies:

Mice were treated with varying doses of anthrax LT as indicated, using a fixed ratio of LF/PA of 1:2.5. LF and PA were resuspended in PBS and injected i.p. into mice in a total volume of 1.0 ml of PBS. As a negative control, selected mice were treated with PBS alone. Mice were sacrificed 3 h after treatment, and spleens were harvested for primary B cell isolation as previously described. Primary B cells were then evaluated for proliferation and IgM production.

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
• Product #176 – Anthrax Lethal Factor (LF), Recombinant, Mutant E687C from B. anthracis
• Product #175 – Anthrax PA 63 – FITC Conjugate

… 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