Bacterial Toxin Research Citations

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

Antigen:

Recombinant PA, produced in an avirulent, non-capsulated, sporulation suppressed Ba host was obtained from List Biological Laboratories, Inc. (Campbell, CA). The PA migrated as a single major band with an apparent molecular weight of 83 000 daltons on 10% polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS). The PA was reconstituted in distilled water and stored aliquoted at 220C. Before use, individual aliquots were thawed and used immediately.

Competitive inhibition:

To determine the specificity of measurements performed by FCMIA, pre-incubation of positive sera, control sera, negative worker sera, and a dilution of AVR414 with PA (competitive inhibition) was performed. Sera (130 ml final volume) were treated with either 80 mg/ml PA (final concentration) or dilution buffer and incubated overnight at 4C. The sera were then centrifuged and the supernatants analysed as outlined above. …

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

Intranasal immunization:

BALB/c mice (n = 5) were intranasally immunized with 10⁹ live S. gordonii DTA₂ cells in 25 µl of PBS containing 10 µg of cholera toxin subunit B (CTB; List Biological Laboratories, Inc., Campbell, Calif.) as the mucosal adjuvant. The method of immunization has been described elsewhere (20, 21). A second group of mice received 10 µg of CTB alone as a control. The animals received additional immunizations on days 21, 33, 47, and 54. The animals were euthanized on day 70, and serum samples and saliva and vaginal wash samples were obtained as described previously (10, 20).

DT neutralization assay:

The DT neutralization assay was performed as described by Miyamura et al. (26) with modifications. Briefly, sera in triplicate were diluted in serial twofold dilutions (starting dilution at 1:2) with growth medium (minimal essential medium with 1% L-glutamine; Invitrogen Life Technologies, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum, 0.01% amphotericin B, 0.3% gentamicin, and 0.2% penicillin G. The diluted samples (12.5 µl) were then transferred to 96-well flat-bottomed Nunclon microplates (VWR International Ltd., Toronto, Canada), and an equal volume of native DT (0.8 ng/ml; List Biological Laboratories) was added. The mixtures were incubated at 37°C for 1.5 h, after which 50 µl of Vero cell suspension (1.2 10⁵ cells/ml) and 150 µl of growth medium were added to each of the wells. The cultures were then incubated in a 5% CO₂ incubator for 5 to 7 days at 37°C and scored with the aid of an inverted microscope for toxicity: +, confluent monolayer; , approximately 50% cell death; , 100% cell death. Neutralization titer was defined as the reciprocal of the dilution that showed complete neutralization (+, confluent monolayer) of DT cytotoxicity.

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

Ags and immunizations:

… CT (230 EU/mg) was obtained from List Biological Laboratories (Campbell, CA). …

Histology:

BALB/c mice were immunized with PBS with or without CTA1-DD or CT. At 24 h after one single, or three intranasal immunization, the heads of the mice with facial skin stripped were severed from the body along the line between the upper and lower jaws. The heads were fixed with 4% formaldehyde, decalcified, and embedded in paraffin. Transverse sections, at about the eye level, were stained by conventional histological procedures using H&E.

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

ELISpot assay for IFN- release – 10 ug/ml of Tetanus Toxoid (List Laboratories, Campbell, CA) or 25ug/ml of Diptheria Toxoid (List Laboratories) 

• Product #191 – Tetanus Toxoid from Clostridium tetani
• Product #151 – Diphtheria Toxoid, from Corynebacterium diphtheriae

ReagentsRecombinant anthrax PA and lethal factor LF were purchased from List Biological Laboratories, Inc. (Campbell, CA) and were stored as 1 mg/ml stock solutions in 1:1 glycerol:water. …

Cytokine induction by LT:

Figure 1:  Relative cytokine levels in the supernatants of RAW264.7 (A) and J774A.1 (B) cells are shown following 24-h treatment with 1 g/ml LF + 2.5 g/ml PA. Supernatants were harvested and tested for cytokine levels by ELISA in duplicate. The y axis values represent the ratio of induction of the various cytokines (gray bars) compared with unstimulated cells (black bars, arbitrarily assigned a value of 1). Shown is a one representative of two cytokine screening experiments. In separate experiments, extracellular levels of IL-1 and IL-18 were measured by ELISA in the supernatants of RAW264.7 (C) or J774A.1 (D) cells stimulated with or without LF (1 g/ml) and/or PA (2.5 g/ml) for 24 h. Bars indicate intraassay standard deviation.

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

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