Bacterial Toxin Research Citations

Materials:

SNAPtide substrate #520 contains the FRET pair o-aminobenzoic acid/2,4 dinitrophenyl (o-Abz/Dnp) and SNAPtidesubstrate Mca/Dnp contains (7methoxy-coumarin-4-yl)acetyl/2,4 dinitrophenyl as its FRET pair. Botulinum Neurotoxin Type A (Product #130A) and the SNAPtidesubstrates are products of List Biological Laboratories.

• Product #520 – SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin

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

•  ELISA assay showing specific binding of BTA to SV2c
•  MagneGST-SV2c capture assay
•  MagneGST-SV2c capture assay with HPLC analysis
•  HPLC peak intensity for cleaved SNAPtideas a function of BTA

Materials:

SNAP Etide550, VAMPtide540, SNYTAXtide560, and SNAPtidesubstrates 520, 521, and 522, and the Botulinum neurotoxin types A, B, C, and E light chains, recombinant (Product #610A, 620A, 625A, and 635A) are products of List Biological Laboratories, Inc.

• Product #550 – SNAP Etide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type E Neurotoxin
• Product #540 – VAMPtide Peptide Substrate (o-Abz/Dnp) Substrate for C. botulinum Type B Neurotoxin
• Product #560 – SYNTAXtide (o-Abz/Dnp) Peptide Substrate for C. botulinum Type C Neurotoxin
• 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 #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant
• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant
• Product #625A – Botulinum Neurotoxin Type C Light Chain, Recombinant
• Product #635A – Botulinum Neurotoxin Type E Light Chain, Recombinant

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

• Fluorimetricassays
• HPLC
• Limit of Detection

LF quantification:

… To achieve the necessary dynamic range and to account for high-sensitivity rapid-response sample analysis, two standard curves were prepared, one for 20 ng/ml, l (low volume [LV]) and the other for 200 ng/ml, l (high volume [HV]) serum volumes. In addition, three quality control samples (QCs) were prepared in human serum pools (Interstate Blood Bank, Memphis, TN) using recombinant LF (List Biological Laboratory, Campbell, CA) and validated. …

PA antigen capture immunoassay:

Microtiter plates (Thermo Labsystems, Franklin, MA) were coated with monoclonal anti-PA antibody AVR1046 obtained by the same methodologies used for anti-LF monoclonal described previously (4) at 1.28 g/ml in 0.01 M phosphate-buffered saline (PBS), pH 7.4. Coated plates were washed with a wash buffer consisting of PBS and 0.1% Tween 20, pH 7.4. Recombinant PA (rPA; List Biological Laboratories, Inc., Campbell, CA) was serially diluted twofold using a 1/25 or a 1/100 dilution of normal rhesus macaque serum (RMS). The rPA dilutions formed a seven-point standard curve from 0.6 to 37.5 ng/ml. …

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

Tracer injections

Several minutes after being given intraperitoneal injections of a cocktail, consisting of 45% ketamine (100mg/ml), 35% xylazine (20mg/ml) and 20% physiological saline at a dose of 0.16 ml/100g of body weight, rats were placed into a Kopf stereotaxic instrument. The skulls were exposed and small bore holes were created to allow selected brain structures to be targeted by filament-containing borosilicate glass pipettes (O.D. – 1.0 mm) pulled to tip diameters of 10–25 μm and containing the retrograde tracers Fluoro-Gold (FG; Fluorochrome, Inc., Englewood, CO; 1% in 0.1M cacodylate buffer, pH 7.4) or cholera toxin B subunit (CtB, List Biological Laboratories, Campbell, CA),

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt

Reagents:

CT and CTB were purchased from List Biological Laboratories (Campbell, CA). …

Immunization:

For peptide immunization, 26106 OT-II splenocytes were i.v. injected into B6 mice, and then recipient mice were i.n. immunized with 40 mg of OVA peptide plus 30 mg of CpG ODN, 0.2 mg of CT, or 2 mg of CT or CTB in PBS. …

Cytokine assays:

Dendritic cells (DCs) were generated from bone marrow of B6 or BALB/c mice by culturing in complete RPMI medium containing 10% FBS supplemented with 10 ng/ml recombinant GM-CSF and IL-4 (R&D, Minneapolis, MN). After 7 days of culture, non-adherent cells were harvested by gentle pipetting, and DCs were enriched by density gradient centrifugation over Percoll medium. After purification, DCs were further incubated with PBS or CT (100 ng/ml) for 3 days and the culture supernatants were harvested at the indicated time points. …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae
• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt

Reagents:

… LPS lyophilized powder (List Biological Labs) was dissolved and diluted in phosphate buffered saline (PBS). As lethality varied among lots of LPS, when a lethal dose of LPS was desired for challenge, each lot of LPS was tested for LD50 before each experiment by injecting nave mice with varying doses of LPS (510 mice per dose group) and assessing survival. The lowest dose that yielded 80100% death was used for the LPS challenge dose in the subsequent experiment.

LPS challenge

All mice were observed every hour for the first 2448 hours following the lethal LPS challenge. Mice were observed for characteristics of LPS toxicity (ocular exudates, diarrhea, increased respiratory rate) as well as moribund status (inability to sustain body temperature, ataxia or tremors, diaphragmatic breathing). In order to assess recovery, mice were given a score from 03 based on ambulation as an indication of overall health status: 0 = unable to walk, 1= take steps to walk, 2 = able to walk quickly but not run, and 3 = able to run/normal ambulation.

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

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

Alcohol administration and intratracheal injection:

Mice received intraperitoneal (i.p.) injection with ethanol [4.0 g/kg; 20% v/v ethanol in sterile phosphate buffered saline (PBS)] or an equivalent volume of PBS. Thirty minutes after i.p. injection, animals were anesthetized with isoflurane for exposure and direct intratracheal (i.t.) administration of 10 g Escherichia coli LPS (List Biological Laboratories; Campbell, CA). n 5 per group for all in vivo experiments.

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

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