Bacterial Toxin Research Citations

Materials:

Recombinant lethal factor (Prod #172), protective antigen (Prod #171), MAPKKide^TM (Prod #530), and MAPKKide^TM Unquenched Calibration Peptide (Prod #539) are products of List Biological Laboratories.

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor
• Product #539 – Unquenched Calibration Peptide for MAPKKide 530 for Anthrax Lethal Factor

Methods – The above materials were used in the following (refer to poster for details):

• HPLC
• Enzymatic activity
• Continuous fluorimetric assays
• LF-PA₆₃ binding assay
• LF enzymatic activity as a function of ZnCl₂

Materials and methods:

Rat LeTx challenge experiments were performed in accordance with protocols approved by the Scripps Institutional Animal Care and Use Committee. Male Fisher 344 rats (180200g; Harlan) were anesthetized with isofluoranes and were inoculated with LeTx mixture through a jugular-vein cannula. LeTx was prepared with 40 mg of PA and 8 mg of LF per rat (List Biological Laboratories). For rats that received sCMG2 and LeTx, sCMG2 was added to the LeTx mixture and was coinjected in a 500-mL volume. Rats recovered from anesthesia within 5 min and were monitored for symptoms of intoxication and death. Statistical analysis was conducted on the basis of Students unpaired t test (Prism, version 4; GraphPad). For rats that died overnight, statistical analysis was based on the last observed postinoculation time point.

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

MATERIALS AND METHODS:

… Recombinant LF and MAPKKide were both purchased from List Biological Laboratories (Campbell, CA). …

Fluorescence Peptide Cleavage Assay:

Cleavage reactions (100 l each) were performed in a 96-well plate. Each reaction contained MAPKKide (4 M) and LF (50 nM) in 20 mM Hepes, pH 7.4, and the small-molecule inhibitor. Kinetics of the peptide cleavage was examined for 30 min by using a fluorescent plate reader at excitation and emission wavelength at 485 and 590 nm, respectively.

The K_m and V_max values of the MAPKKide cleavage by LF were determined at 25C by using the same experimental condition described above for the fluorescence screening assay but with increasing MAPKKide concentrations (2, 3, 5, 8, and 10 M). The K_i and K_m(app) were calculated at a fixed 10 M inhibitor concentration. All constant values were definitely evaluated by fitting the data to the LineweaverBurk plot.

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

Materials:

SNAPtide substrates (product #520 and #521), SNAPtide Unquenched Calibration Peptides (product #528 and #529), botulinum neurotoxin type A (product #130A), botulinum neurotoxin type A light chain, recombinant (product #610A ), botulinum neurotoxin type B light chain, recombinant (product #620A ) and VAMPtide (product #540) are all products of List Biological Laboratories, Inc.

• 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 #528 – Unquenched Calibration Peptide for SNAPtide 521 Substrate for C. botulinum Type A Neurotoxin
• Product #529 –  Unquenched Calibration Peptide for SNAPtide 520 Substrate for C. botulinum Type A Neurotoxin
• Product #130 – Botulinum Neurotoxin Type A from Clostridium botulinum
• Product #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant
• Prouct #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant
• Product #540 –  VAMPtide Peptide Substrate (o-Abz/Dnp) Substrate for C. botulinum Type B Neurotoxin

Methods – The above materials were used in the following (refer to poster for details):

• Fluorimetric assays
• Zinc titration
• Limit of Detection (LOD)
• VAMPtide

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