Bacterial Toxin Research Citations

… PA63 and LF were purchased from List Biological Laboratories (Campbell, CA). …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

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).

In vivo intoxication experiments:

Lyophilized recombinant PA, LF, and mutant LF (E687C) were purchased from List Biological Laboratories, Inc. (Campbell, CA) and reconstituted in sterile water (stock concentration: 1 mg/mL in 5 mM HEPES, 50 mM NaCl, pH 7.5). Anthrax LT was administered with a fixed ratio of PA/LF of 2.51 by weight (molar ratio: 2.71). Unless otherwise indicated, mice were injected intravenously with 200 g PA/80 g LF (C57BL/6J) or 100 g PA/40 g LF (BALB/c), administered in a total volume of 0.3 mL of PBS. As a negative control, selected mice received the same volume of PBS alone. …

Some experiments involved the concomitant administration of broad-spectrum antibiotics with anthrax LT. Both antibiotics were administered subcutaneously beginning the day of LT treatment (amoxicillin: 100 mg/kg every 8 hours, and gentamicin 16 mg/kg one time per day, Sigma, St. Louis, MO). Antibiotic therapy was maintained until the animal succumbed to toxemia or until the termination of the experiment.

• Product #176 – Anthrax Lethal Factor (LF), Recombinant, Mutant E687C from B. anthracis

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

Functional Assays :

Anthrax LF was obtained from List Biological Laboratories, Inc. …

Briefly, freshly prepared LF at a final concentration of 1 g/ml (10 nm) was incubated at 37 C for 30 min with a 2-fold dilution series of defensin in 20 mm HEPES buffer containing 1 mm CaCl2 and 0.5% Nonidet P-40, pH 7.2. 20 l of LF substrate, …

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).

Nasal vaccination:

Mice were anesthetized with isourane before vaccination. Vaccines were administered intranasally in 10 ml (5ml per nostril). Nonchimeric mice were vaccinated on days 0, 7, and 21. Chimeric mice were vaccinated on days 0, 7, 21, and 42. For each chimeric study replicate, mice were divided into groups of 4 to 8. Vaccine groups included PBS alone, recombinant LF (rLF) alone (#172; List Biological Laboratories Cambell, CA; 4 endotoxin units [EU]/mg), rLF + 4 mg IL-1a (400-ML; 0.00144 EU/mg; R&D, Minneapolis, MN), and rLF + 1 mg cholera toxin (CT) (EU/mg unknown; List #100B). For three of the four Il1r1 chimeric mouse experiments, mice were vaccinated with 25 mg LF for the day 0, 7, and 21 immunizations and 50 mg LF on day 42. For the remaining experiment, mice were immunized with 50 mg LF each time. The calculated endotoxin delivery per mouse was 0.20 (LF alone) to 0.22 EU (LF + IL-1a). Data from each group for all repeat experiments were combined and analyzed together. Mice vaccinated with LF + CT served as a positive control, and LF + CT-vaccinated mice responded similarly in all four chimeric groups.

• Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis)
• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Nasal vaccination:

Mice were anesthetized with isourane before vaccination. Vaccines were administered intranasally in 10 ml (5ml per nostril). Nonchimeric mice were vaccinated on days 0, 7, and 21. Chimeric mice were vaccinated on days 0, 7, 21, and 42. For each chimeric study replicate, mice were divided into groups of 4 to 8. Vaccine groups included PBS alone, recombinant LF (rLF) alone (#172; List Biological Laboratories Cambell, CA; 4 endotoxin units [EU]/mg), rLF + 4 mg IL-1a (400-ML; 0.00144 EU/mg; R&D, Minneapolis, MN), and rLF + 1 mg cholera toxin (CT) (EU/mg unknown; List #100B). For three of the four Il1r1 chimeric mouse experiments, mice were vaccinated with 25 mg LF for the day 0, 7, and 21 immunizations and 50 mg LF on day 42. For the remaining experiment, mice were immunized with 50 mg LF each time. The calculated endotoxin delivery per mouse was 0.20 (LF alone) to 0.22 EU (LF + IL-1a). Data from each group for all repeat experiments were combined and analyzed together. Mice vaccinated with LF + CT served as a positive control, and LF + CT-vaccinated mice responded similarly in all four chimeric groups. …

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Experimental protocols:

After completion of the surgical procedures, anesthetized rats were allowed to stabilize for 60 min before initiation of experimental protocols. As described by Cui et al. (4, 6), rats received LeTx [LF (100 g/kg) + PA (200 g/kg) infused at 0.5 ml/h] or vehicle (PA + PBS with 1% BSA or PBS with 1% BSA infused at 0.5 ml/h). LF and PA are recombinant proteins prepared from B. anthracis and purchased from List Biological Laboratories. Experiments were completed in intact and SAD rats. LeTx or vehicle was infused intravenously via a femoral venous catheter, and the dose of LeTx was chosen on the basis of the time course of physiological responses to intravenous LeTx infusions as reported by Cui and colleagues (6). MAP, HR, and SND were recorded continuously during intravenous LeTx or vehicle infusions. The maximum infusion duration was 6 h; however, because of LeTx-induced reductions in arterial blood pressure, the majority of experiments were terminated before 6 h. Additional experiments involved the intravenous infusion of sodium nitroprusside (SNP) at a dose (20 g/kg) that reduced basal levels of MAP to 5060 mmHg. At the end of experiments, rats were euthanized by an overdose of methohexital sodium (150 mg/kg iv).

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

… Purified recombinant protective antigen (rPA) was obtained from three different sources; 1) Dr. Stephen H. Leppla, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD; 2) List Biologicals, Campbell, CA or …

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

Reagents:

… The PA ligand was purchased from List Laboratories (Campbell, USA). …

Surface modification:

… After washing with HBS for 2 min at a flow rate of 30 m/min, 100 g/ml of PA was introduced and incubated for 30 min with a slow flow rate of 3 l/min for covalent binding to the SAM layer. …

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

Chemicals and Reagents:

PA and LF were purchased from List Biological Laboratories.

Toxin Treatment and Cell Viability Assays:

Mouse macrophages were treated with toxin for 4 h and human B lymphocytes for 48 h. Determination of macrophage viability was done by MTT assay as described in refs. 31, 32, whereas lymphocyte viability was determined by Resazurin (Invitrogen) fluorescence, as described by the manufacturer. Each data point shown in Figs. 1 and and22 represents the average and SD of results from three wells. Cell viability is shown as the percentage of survivors obtained relative to treatment by PA alone (100%). LD₅₀ was calculated for each cell line and normalized to account for variation in toxin potency from the two different lot numbers used, as determined in five and three independent LD₅₀ measurements of a control cell line for the first and second lots, respectively.

Immunofluorescence Microscopy:

PA protein was labeled with Alexa Fluor 647 using the protein labeling kit (A20173; Molecular Probes). We determined by MTT assay that such labeling did not affect the ability of PA/LF to kill macrophages. Fresh serum-free IMDM media was added to cells that contained 1 g/mL PA/Alexa Fluor 647. Cells were incubated for 20 min either at 4 C for PA-binding analysis or at 37 C for determination of PA internalization.

Biochemical Assay of PA Binding and Internalization:

Three million macrophage cells were exposed to 1 g/mL of PA at 4 C for 1 h for binding assay or at 37 C for 20 min for internalization assay. Cells were then washed with PBS solution three times and lysed in RIPA buffer containing a protease inhibitor mixture (Roche). Western blot analysis was performed using mouse monoclonal anti-PA antibody PA (C3) sc-52129 (Santa Cruz Biotechnology), or mouse antiactin monoclonal antibody (Sigma-Aldrich). Chemiluminescence of bands and their relative intensities were revealed using a VersaDoc 1000 instrument (Bio-Rad).

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

… For direct ELISA analysis of the peptide binding affinity to a recombinant PA protein, 83 kDa PA (PA 83 ; List Biological Laboratories, Campbell, CA, USA) was labeled directly with 44 kDa horseradish peroxidase enzyme (HRP) using EZ-Link plus activated peroxidase …

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