Bacterial Toxin Research Citations

PGRP-S secretion:

Granulocyte-enriched populations of bovine peripheral leukocytes were purified as described above and suspended to 1.2 10⁷ cells/ml in HBSS (137 mM NaCl, 5.6 mM glucose, 5 mM KCl, 4 mM NaHCO₃ 1 mM CaCl₂, 0.5 mM MgCl₂, 0.4 mM KH₂PO₄, 0.4 mM Na₂HPO₄, and 0.4 mM MgSO₄ (pH 7.4)). Aliquots of the cell suspension were incubated for 60 min at 37C in a final volume of 500 l containing one of the following stimulants: 100 nM PMA, 20 g/ml lipoteichoic acid (LTA) from Bacillus subtilis, 160 g/ml muramyl dipeptide (MDP) (Sigma-Aldrich), 100 g/ml LPS from Staphylococcus typhimurium (List Biological Laboratories), …

• Product #225 – LPS from Salmonella typhimurium

… 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:

Botulinum Neurotoxin Type A (Prod #130A) and the recombinant light chain of the Type A neurotoxin (Prod #610A) are products of List Biological Laboratories.

FVIII and Tetanus Toxoid Immunizations:

… Two of the 800 cGy-irradiated mice (mice #26 and #27) and four naive E16 mice were immunized with tetanus toxoid (List Biological Laboratories, Inc., Campbell, CA, USA) 3 weeks after the final FVIII injection as follows: 4 g tetanus toxoid was mixed with 1 mg Al (OH)₃adjuvant (Alhydrogel 2%; Accurate Chemical & Scientific Corporation, Westbury, NY, USA), diluted in sterile saline to a total volume of 108 l and injected intraperitoneally. Two weeks later, sera from these mice was assessed for the presence of anti-tetanus toxoid antibodies by ELISA (described below).

• Product #191 – Tetanus Toxoid from Clostridium tetani

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

Materials:

SNAPtide substrates (product #520 and #521), SNAPtide Unquenched Calibration Peptides (product #528 and #529), botulinum neurotoxin type A (product #130A), botulinum neurotoxin type A light chain, recombinant (product #610A ), botulinum neurotoxin type B light chain, recombinant (product #620A ) and VAMPtide (product #540) are all products of List Biological Laboratories, Inc.

• Product #520 – SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin
• Product #521 – SNAPtide Peptide Substrate (FITC/DABCYL) for C. botulinum Type A Neurotoxin
• Product #528 – Unquenched Calibration Peptide for SNAPtide 521 Substrate for C. botulinum Type A Neurotoxin
• Product #529 –  Unquenched Calibration Peptide for SNAPtide 520 Substrate for C. botulinum Type A Neurotoxin
• Product #130 – Botulinum Neurotoxin Type A from Clostridium botulinum
• Product #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant
• Prouct #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant
• Product #540 –  VAMPtide Peptide Substrate (o-Abz/Dnp) Substrate for C. botulinum Type B Neurotoxin

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

• Fluorimetric assays
• Zinc titration
• Limit of Detection (LOD)
• VAMPtide

Mice and reagents:

Five-week-old female C3H/HeJ mice purchased from the Jackson Laboratory (Bar Harbor, Me) were maintained on peanut-free chow under specific pathogen-free conditions according to standard guidelines for the care and use of animals …. Reagents were purchased from the following sources: cholera toxin, List Biological Laboratories, Campbell, Calif; …

Intragastric peanut sensitization, challenge, and FAHF-2 treatment:

Mice were sensitized intragastrically with ground peanut (10 mg/ mouse) and cholera toxin (20 mg/mouse) weekly for 3 weeks as previously described,14 then treated intragastrically with FAHF-2 (20 mg/mouse in 0.5 mL water) twice daily for 7 weeks (weeks 3-9). …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae