Bacterial Toxin Research Citations

We next tested the ability of RDI to neutralize LeTx containing an antibody-resistant form of PA (PA^L685A) in a rat model system. Male Harlan Sprague-Dawley rats (180 to 200 g; Harlan, Indianapolis, IN) were anesthetized with isofluoranes and inoculated with 500 l LeTx mixture by intravenous injection in the tail vein. The anthrax LeTx was prepared for each group by mixing 12 g LF (List Biological Laboratories, Campbell, CA) with 40 g PA (List Biological Laboratories, Campbell, CA) …

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 the procurement of positive control hyperimmune anti-PA and anti-LF serum for use in the western blotting and as positive controls for the LeTx neutralization assays, rabbits were immunized with 250 g of either soluble PA83 or LF in an emulsion with CFA (List Biological Laboratories, Campbell, CA). Rabbits were then boosted three times at 23-week intervals with 125 g of the respective immunogens in an emulsion with incomplete Freund’s adjuvant …

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

Samples of LF used for comparison were purchased from List Biological Laboratories (Campbell, CA).

Results – Toxicities of LF proteins produced from avirulent B. anthracis strains:

… B. anthracis strains cured of the pXO1 and pXO2 virulence plasmids [18]. LF expressed from pSJ115 is here termed LF-HMA to denote the presence of the two residues His-Met (HM) added at its N-terminus due to the cloning manipulations. This expression system is licensed to List Biological Laboratories (Campbell, CA), and the LF sold by them is therefore also LF-HMA. …

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

Microinjection of Toxin Proteins and Use of Inhibitors:

… LF was purchased from List Laboratories, Inc. …

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

Quantification of PA₈₃ bound to chimeras 206 and 264:

Recombinant PA₈₃ (List Biological Laboratories) in 5 mM Hepes, 50 mM NaCl (pH 7.5) was mixed with purified chimeras 206 and 264 in 50 mM Hepes (pH 7.5) in a ratio of 180:1 (equimolar amounts of PA₈₃and VWA domains). Following incubation for 20 min at room temprature, an aliquot from each of the samples was removed and stored at 20 C pending analysis. The remainder of the samples was transferred to an ultracentrifuge tube and underlayed with a 30% (w/w) sucrose cushion in 50 mM Hepes (pH 7.5). Complexes of chimeras decorated with PA₈₃ were pelleted by centrifugation at 200,000g for 45 min. …

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

…Recombinant B. anthracis lethal factor (rLF) and protective antigen (rPA) were acquired from List Biological Laboratories, Inc. (Campbell, CA) …

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

The following FRET peptides have been designed at List Biological Laboratories as substrates for the botulinum toxin enzymes. SNAPtide, Product #520 and #521 (U.S. Patent, No. 6,504,006 ) is readily recognized and cleaved by the Botulinum toxin type A (BTA), Product #130, and the BTA light chain (LcA), Product #610A. One of the substrates, Product #520, contains an oAbz/DNP FRET pair and the other, Product #521, a FITC/DABCYL FRET pair. Representative data are given below. VAMPtide, Product #540 is readily recognized and cleaved by the Botulinum toxin type B light chain (LcB), Product #620A. This FRET substrate contains an oAbz/DNP FRET pair. SNAP Etide, Product # 550 is readily recognized and cleaved by the Botulinum toxin type E light chain (LcE), Product #635A. This FRET substrate contains an oAbz/DNP FRET pair.

• 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 #130 – Botulinum Neurotoxin Type A from Clostridium botulinum
• Product #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant
• Product #540 – VAMPtide Peptide Substrate (o-Abz/Dnp) Substrate for C. botulinum Type B Neurotoxin
• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant
• Product #550 – SNAP Etide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type E Neurotoxin
• Product #635A – Botulinum Neurotoxin Type E Light Chain, Recombinant

(LF) is the enzymatic component of anthrax lethal toxin which specifically cleaves the MAPK-kinase proteins. LF is also an ideal target for therapeutic inhibitors. FRET substrates for LF, MAPKKide, Product #530 and 531, have been designed at List Biological Laboratories. One of the substrates, Product #530, contains an oAbz/DNP FRET pair and the other, Product #531, a FITC/DABCYL FRET pair. Product #531 is especially well suited for high throughput screening for the IC50 and inhibitor modality of potential inhibitors.  …

• Product #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor
• Product #531 – MAPKKide Peptide Substrate (DABCYL/FITC) for Anthrax Lethal Factor

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