Bacterial Toxin Research Citations

Recombinant protein reagents:

Recombinant anthrax toxins, rPA, rLF, or rEF (List Biological Laboratories, Campbell, CA) were stored frozen in 1 mg/ml aliquots as recommended by the supplier. …

Protective antigen, lethal factor, and edema factor ELISAs:

Antibody specificity for toxin components was determined using standard ELISAs. High bind plates (Costar 3369) were coated overnight with 1 g/well of rPA, rLF, or rEF. …

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

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Synthesis and Folding of Defensins:

… Recombinant LF was purchased from List Biological Laboratories, Inc. …

LF Activity Inhibition by l-Ala17-HNP1 Was Less Than Wild-type HNP1:

The enzyme activity of LF in the presence of HNP1 or l-Ala17-HNP1 was assessed using a method adapted from Kaufmann and colleagues (30). Fig. 7 shows that the inhibitory activity of l-Ala17-HNP1 as measured by the concentration that gave 50% inhibition (IC50) differed slightly more than 2-fold compared with HNP1. By contrast, d-Ala17-HNP2 (HNP1 numbering) was equally active compared with HNP2. The d-Ala17 mutation was incorporated into HNP2 instead of HNP1 to conform to the previous study (18), but because HNP1 and HNP2 vary only by the deletion of a single Ala residue at the N terminus, they should be comparable with one another with respect to the internal Gly17 residue. LF inhibition is impaired only by the Gly17 to l-Ala17 mutation but not the Gly17 to d-Ala17 mutation, pointing to a local structural defect as the culprit.

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).

Animal models of anthrax infection:

… Groups of female Hartley guinea-pigs (250-280 g) were immunized by intramuscular injection into the hind leg with 0.2 ml of 50 g of PA (List Biological Laboratories, Inc.), D4 or PDGA-D4 absorbed to 25% aluminum hydroxide (Alhydrogel) on days 0, 14 and 28. …

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

… The B. anthracis proteins, protective antigen (PA), edema factor (EF), and lethal factor (LF) were purchased from List Biological Laboratories, Inc. and required HRP labeling prior to analysis. …

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

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

… Recombinant Bacillus anthracis lethal factor (LF) and Clostridium botulinum neurotoxin light chain A (BoNTALC) were obtained from List Laboratories (Campbell, CA; 169A and 610A, respectively). …

Protease Inhibition Assay:

Protease inhibition assays were performed as described
previously (25), but with modifications. Biotinylated green
fluorescent protein (GFP) protease substrates for LF, BoN-
TALC, and a protease-resistant substrate (pinpointGFP)
were prepared and loaded on streptavidin microspheres as
described previously (14,25,26). Additions to wells were in
sequence as follows: (1) 4 lL protease buffer; (2) 2 lLofa
mixture of 1.5 lM LF and 5 nM BoNTALC in protease
buffer; …

• Product #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant

List Labs Product #169A (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Lyophilized) has been discontinued; however, Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Liquid) is available for purchase.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

… 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