Bacterial Toxin Research Citations

Reagents:

Recombinant B. anthracis LF and PA (rPA) were acquired from List Biological Laboratories (Campbell, CA, USA). …

Immunization of rabbits and sheep:

Rabbits were immunized s.c. with rPA mixed with Freund’s complete adjuvant (FCA) initially, and with Freund’s incomplete adjuvant (FIA) at the time of boosting 3 and 6 weeks later. Rabbits were bled 1 week after the last immunization. …

Cytotoxicity neutralization test:

LeTx (LF=16 ng ml¹ or 64 ng ml¹; PA=500 ng ml¹) was pre-incubated in DMEM with varying concentrations of the tested antibody for 1 h at 37C in a 5% CO₂atmosphere. …

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

Cytotoxicity assays:

Equal numbers of wild-type or FS-transfected cells were suspended in 400 l of replete medium, and 2.5 10⁴ cells were aliquoted into 24-well dishes. The following day, increasing dilutions of Stx1 (15,000 to 1.5 ng/ml; List Laboratories) were added. Cells were then exposed to toxin dilutions in complete medium for 48 h in order to maximize toxin effect. Similar results were seen with a 4-h incubation in the presence of toxin, though the degree of protein synthesis inhibition was less profound among all cell types (data not shown). …

• Product #161 – Shiga Toxin 1 from Escherichia coli

Reagents:

Escherichia coli K12 D31m4 (Re) LPS and lipid A were purchased from List Biological Laboratories. Control experiments with this LPS preparation did not exhibit any stimulating activity on peritoneal macrophages from TLR4-deficient mice at the concentrations used in this study. …

Cell Stimulation Assays:

Cells were seeded on culture plates coated with 2 mg/ml rat tail collagen type 1 diluted 1:100 in ethanol/water (60:40 vol/vol) and incubated for 6 d with medium changes every second day (10, 11). LPS or lipid A was vortexed, sonicated for 15 min, and added to the cells at the appropriate concentration. For the determination of luciferase activity, cells were stimulated for 2 h, washed with cold PBS, and incubated for 10 min in lysis buffer (Promega). Luminescence was recorded after the addition of substrate (Promega) using a TD 20/20 Luminometer (Turner Designs Instruments). If not stated otherwise, m-IC_cl2 cells were exposed to the various drugs to be tested for 30 min before LPS stimulation. Cell viability was monitored using the CytoTox 96 cytotoxicity assay (Promega). …

• Product #302 – LPS from Escherichia coli K12, D31m4 (Re)
• Product #401 – LIPID A monophosphoryl from Salmonella minnesota R595

Materials:

… Anthrax protective antigen was purchased from List Biological Laboratory (Campbell, CA), …

Purification of EF, EF3, CyaA-N, and CaMEF3 and CyaA-N, the catalytic domains of EF and CyaA, respectively, as well as calmodulin were purified as described previously (37,38). To express edema factor that has a hexahistidine tag substituted for its leader peptide (amino acids 133) and can be delivered by anthrax-protective antigen into host cells, a plasmid, pProEx-H6-EF, was constructed as follows. The 3.2-kb EcoRI-XhoI fragment was excised from pSE42 (kindly provided by S. Leppla, National Institutes of Health) and inserted into pBluescript. A NotI site was then introduced at the sequence encoding amino acids 3234 of EF by site-directed mutagenesis, and the mutation was confirmed by DNA sequencing. 

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

Materials:

Tetanus toxin was provided by List Biological Laboratories Inc. (Campbell, CA, USA). …

Results:

… As can be seen in Fig. 1, treatment with a moderate TeTx concentration of 1.0 nM was able to block the specific [3H]5-HT uptake in P2 fractions. On the other hand, the growth factors used, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), which are endogenous agonists of some tyrosine-kinase receptors, were unable to produce the same effect at the indicated concentrations in Fig. 1. On the contrary, the growth factors tested produced significant increases in [3H]5-HT uptake (127.2% of Control with 200 nM EGF, 151.2% with 200 nM bFGF, and 138.5% with 200 nM NGF). Co-application of the neurotoxin (1 nM) with each one of the indicated growth factors was able to produce a progressive, dose-dependent reversion of the TeTx effect. …

• Product #190 – Tetanus Toxin from Clostridium tetani

PA-Binding Studies:

PA binding to CHO-R1.1 cells that expressed CMG2⁴⁸⁹-EGFP or ATR/TEM8 sv2-EGFP was assessed by flow-cytometric analysis; 10⁶ cells of each type were incubated with 50 nM PA (1 h, 4C), washed with PBS, incubated with a polyclonal PA-specific rabbit antiserum (9) (1:500 dilution, 1 h, 4C), washed with PBS again, and then incubated with an allophycocyanin-conjugated anti-rabbit antibody (Molecular Probes; 1:2,000 dilution, 1 h, 4C). ELISAs were performed by binding 0.3 g of PA83 (List Biological Laboratories, Campbell, CA)/100 l of TBS in a well of a MaxiSorp plate (Nalge). Samples were then treated with TBST solution (TBS containing 3% BSA and 0.05% Tween 20), washed with TBST, and incubated with 0300 ng of purified CMG2^VWA/I-MycHis/100 l TBST in the absence or presence of 1 mM MgCl₂, MnCl₂, CaCl₂, or ZnCl₂. The samples were then washed with TBST and incubated with a horseradish peroxidase-conjugated anti-His antibody (1:2,000 dilution; Santa Cruz Biotechnology). All incubations for ELISAs were performed for 1 h at room temperature. The levels of bound antibody were then measured by using Supersignal ELISA Pico chemiluminescent substrate (Pierce) and a luminometer (Victor V, Wallac).

Intoxication Assays:

Cell viability assays (WST-1 assay; Roche Molecular Biochemicals) were performed in triplicate by incubating 5,000 cells of each type for 30 h with 2 10¹⁰ M LF_N-diptheria toxin A chain (DTA) and varying concentrations of PA (10¹² to 10⁸ M; List Biological Laboratories) or no PA (100% viability control). The inhibition assays were performed as above with different amounts of CMG2^VWA/I-MycHis (01,000 ng/100 l) added to 10⁹ M PA and 10¹⁰ M LF_N-DTA before this mixture was added to cells. The IC₅₀ was determined by regression analysis (prism, GraphPad, San Diego).

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

Materials:

… The purified recombinant anthrax lethal factor (LF) and both MAPKKide substrates are products of List Biological Laboratories.

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

Materials:

ReLPS from Salmonella Minessota Re595 was purchased from List Labs (CA, USA). …

Repurification of LPS prepration:

Commercial LPS was resuspended in endotoxin-free water containing 0.2% triethylamine followed by vortexing. LPS was repurified using a modified phenol-water extraction procedure followed by ethanol precipitation as described previously ( Tapping et al., 2000; Hirschfeld et al., 2000; Manthey and Vogel, 1994). Recovery of LPS was determined by 3-deoxy-D-manno-octulosonic acid assay.

TNF induction:

Human monocytes (510⁵) were mixed with 50 l of serial dilutions of LPS in the presence of 1% HPS in serum free medium. After 2.5 hours, the supernatant was collected and analysed for TNF- using an enzyme-linked immunosorbent assay (Research Diagnostics Inc). For inhibition experiments, cells were treated with either 60 g/ml nystatin or 10 mM MCD for 10 minutes prior to LPS stimulation.

• Product #304 – LPS from Salmonella minnesota R595 (Re)

Immunization protocol:

A standard nasal immunization protocol was used in this study [12]. C57BL/6 mice were nasally immunized to both nostrils on days 0, 7 and 14 with 25 g of diphtheria toxoid (DT) (1050 Lfu/mg) kindly provided by the Biken Foundation (Osaka University) either alone or combined with 5g of the LPS-free form of mCT or 0.5g of nCT (List Biologic Laboratories Inc.) as mucosal adjuvants.

Detection of neutralizing Abs by use of diphtheria toxin-specific Vero cell assay:

The cytotoxicity induced by the diphtheria toxin (List Biological Laboratories Inc.) in Vero cell cultures was used to measure the toxin-specific neutralizing titers of the Abs induced by the nasal vaccine.

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae
• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

Materials:

TeTx was provided by List Biological Laboratories, Inc. (Campbell, CA, USA). …

Results:

• TeTx, like TPA, induces [3H]5-HT uptake inhibition in rat-brain synaptosomes
• TeTx induces dose-dependent translocation of PKC isoforms
• TeTx induces both PLC activation and PLCQ1 phosphorylation at Tyr residues via tyrosine kinase receptors (TyrKRs)
• TeTx, like TPA, induces protein phosphorylations in Ser residues and specic SERT phosphorylation
• Bisindolylmaleimide-1 (BIM-1) blocks the TeTx and TPA induction of SERT phosphorylation

• Product #190 – Tetanus Toxin from Clostridium tetani