Bacterial Toxin Research Citations

… Briefly, 10 ng STa-ovalbumin conjugates (provided by Dr. DC Robertson at Kansas State University) was used to coat each well of Costar plates (Corning Inc., Corning, NY) to titrate anti-STa antibodies, and 100 ng LT (List Biological Laboratories, Inc., Campbell, CA) was coated to each well of 2HB plates (Thermo Scientific, Rochester, NY) to titrate anti-LT antibodies. …

Lethal Toxin (LT) is PA + LF:

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

Lethal toxin and signaling inhibitors:

Recombinant protective antigen and wild-type lethal factor or lethal factor mutant (E687C) were obtained from List Biologicals (Campbell, CA) …

In vitro lethal toxin assay with Rag1^-/- splenocytes or human tonsillar lymphocytes:

100,000500,000 splenocytes per well were incubated in a round bottom 96 well plate in IMDM media supplemented with 10% FBS, 100 U/ml pencillin, 100 U/ml streptomycin, IL-2 (20 ng/ml) and IL-7 (10 ng/ml). Human tonsillar lymphocytes (2×10⁶) were plated in 200 l RPMI media supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, non-essential amino acids, sodium pyruvate, IL-2 (20 ng/ml), IL-7 (10 ng/ml) and IL-1 (20 ng/ml). For initial lethal toxin experiments splenocytes/tonsillar lymphocytes were cultured in media containing in 0.1% serum to minimize the effect of serum on lethal factor enzyme activity. After the initial period of lethal toxin treatment (3 hrs), serum was replenished to a full concentration of 10%. Cells after toxin treatment were stimulated with recombinant mouse or human IL-23 (50 ng/ml) (eBioscience) for 18 hrs. Cell supernatants were harvested by centrifugation at 1,500 rpm for 5 minutes and then used for the measurement of IL-22.

In vitro lethal toxin assay with MNK-3 cells:

MNK-3 cells (an ILC3 cell line) were cultured in MNK-3 media (DMEM (High glucose), 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM Glutagro, 1 mM sodium pyruvate, 10 mM HEPES, 55 M -mercaptoethanol, 5 g/ml gentamicin, 10 ng/ml IL-7 and 10 ng/ml IL-15). For lethal toxin experiments MNK-3 cells were cultured in media containing 10 ng/ml IL-7. Cells were treated with lethal toxin for 2 hrs followed by IL-23 stimulation for 6 hrs. Cell lysates were analyzed for RNA and supernatants were analyzed for secreted IL-22 by ELISA.

Lethal Toxin (LT) = PA + LF

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

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

… Given the important role of pH change in the anthrax toxin internalization process, the current study is designed to address the disparity.

Material and Methods:

PA63 was purchased from List Biological Laboratories, Inc. (Campbell, CA). …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

Cells and Reagents:

… Lyophilized recombinant PA and LF were purchased from List Biological Laboratories, Inc. (Campbell, CA), and reconstituted in sterile water to make stock solutions with final concentrations of 1 mg/mL. …

Cell Proliferation Analysis:

Eighty thousand Hepa1c1c7 cells were added into individual wells of 12-well plates. Six replicates were used for each group. The cells were treated with or without LT for 48 h. The cell number in each well was counted after the treatment using a TC20 automated cell counter (BioRad, Hercules, CA). LT-induced cell proliferation inhibition was calculated using the following formula: …

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

Toxin neutralization assay (TNA):

An in vitro toxin neutralization assay (TNA) was performed using a MTT [3-(4.5-dimethylthiazol-2-yl)2.5-diphenyltetrazolium bromide] in a colorimetric cell viability assay with the J774A.1 macrophage cell line as previously described by Hering et al. [18] with slight modifications by Ndumnego et al. [17]. Briefly, serial diluted goat serum was incubated with PA and LF (List Biological Laboratories, USA) at concentrations of 500 and 400 ng/mL respectively before addition to overnight incubated macrophage cells. …

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

… ELISAs were performed as described previously (58). Briefly, 96-well plates were coated at 4C overnight with 1 g/well of recombinant PA or EF (List Biologicals, Campbell, CA). …

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

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Materials and Methods

Anthrax lethal factor (Product #169), and the chicken IgY polyclonal anti-LF antibody (Product # 769A) are products
of List Biological Laboratories, Inc. The C8 Starwell Maxi Nunc-Immuno Module Plates (cat# 441653) used for LF
antibody coating and the dimethyl sulfoxide (DMSO) (cat # TS-20684) were purchased from ThermoScientific. The
96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning (cat # 3991).
Bovine plasma (cat # 7310806) was purchased from Lampire Biological Laboratories.

Sample Preparation: Stock solutions of the fluorogenic substrate, MAPKKide® Plus, were 1.25 mM in DMSO
based on the peptide content determined by elemental analysis. The substrate was diluted in assay buffer:
20 mM HEPES, pH 8.0 containing 0.1% Tween-20. For the microplate assays, the LF was dissolved in neat bovine
plasma and diluted 1:10 in assay buffer. For the HPLC assays, the LF was added to neat bovine plasma and not
diluted.

LF Activity Assays:

Microplate reader: Assays were performed on a SPECTRAmax GEMINI XS fluorescence microplate reader
(Molecular Devices). The cleavage reaction was initiated by addition of the substrate, MAPKKide® Plus. The
concentration was optimized to minimize background fluorescence while maintaining measureable cleavage. For
all experiments the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 or 6 hours followed
by an additional 18 to 18.5 hr overnight incubation at ambient temperature. The excitation wavelength was set to
368 nm and emission to 452 nm with a cutoff filter at 435 nm.

HPLC: The C8 Starwell Maxi Nunc-Immuno Module Plates were coated with 150 μl of a 10 μg/ml solution of
a chicken affinity purified polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A). Plates were incubated
with the IgY overnight at 2-8°C and washed three times with 0.1M Glycine-HCl, pH 2.5. This wash was included to
liberate residual LF retained after the affinity purification of the antibody and was necessary to minimize the background. After 6 washes with PBS containing 0.05% TWEEN-20 (PBST), the anti-LF coated wells were exposed to
300 μl of a series of LF concentrations in neat plasma. The plates were incubated at 22°C for 2 hours. Plates were
then washed 6 times with PBST and 250 μl of 1.25 μM MAPKKide® Plus was added. The reaction was allowed to
proceed for 2, 3.5, and 5 hours at 37°C and overnight at ambient temperature. At each time point 200 μl of the
reaction mixture was removed from replicate wells and placed in HPLC sample vials.

HPLC was performed using a Zorbax Eclipse Plus C18 reverse phase column, 4.6 x 150 mm (Agilent) and a guard
column containing the same resin in a Varian ProStar HPLC system (Agilent). Solvent A was 0.1% TFA in water and
solvent B was 0.1% TFA in acetonitrile. The 16 minute HPLC method was as follows: 25% B for 0.75 minutes;
25 to 45% B in 4.75 minutes; 45 to 100% B in 0.75 minutes; 100% B for 3.75 minutes; 100 to 25%B in 0.67 minutes
and 5.34 minute equilibration with 25% B. The column effluent was monitored using an Hitachi fluorescence detector
with excitation set to 350 nm and emission at 450 nm to detect the free coumarin fluorophore cleaved from
MAPKKide® Plus. The injection volume was 20 µl. The 7-amido-4-methylcoumarin (AMC) peak retention time
was 4.8 minutes.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #769B – Anti-Lethal Factor from B. anthracis (Chicken lgY)
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

Materials and Methods

Anthrax lethal factor (Product #169), and the chicken IgY polyclonal anti-LF antibody (Product # 769A) are products
of List Biological Laboratories, Inc. The C8 Starwell Maxi Nunc-Immuno Module Plates (cat# 441653) used for LF
antibody coating and the dimethyl sulfoxide (DMSO) (cat # TS-20684) were purchased from ThermoScientific. The
96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning (cat # 3991).
Bovine plasma (cat # 7310806) and sheep plasma (cat# 7319006) were purchased from Lampire Biological
Laboratories. The milk (2% Lucerne) came from the local market.

Sample Preparation: Stock solutions of the fluorogenic substrate, MAPKKide® Plus, were 1.25 mM in DMSO
based on the peptide content determined by elemental analysis. The substrate was diluted in assay buffer:
20 mM HEPES, pH 8.0 containing 0.1% Tween-20. For the microplate assays, the LF was dissolved in neat bovine
plasma and diluted 1:10 in assay buffer. For the HPLC assays, the LF was added to neat bovine, sheep plasma or
2% milk without dilution.

LF Activity Assays:
Microplate reader: Assays were performed on a SPECTRAmax GEMINI XS fluorescence microplate reader
(Molecular Devices). The cleavage reaction was initiated by addition of the substrate, MAPKKide® Plus. The
concentration was optimized to minimize background fluorescence while maintaining measureable cleavage. For
all experiments the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 or 6 hours followed
by an additional 18 to 18.5 hr overnight incubation at ambient temperature. The excitation wavelength was set to
368 nm and emission to 452 nm with a cutoff filter at 435 nm.

HPLC: The C8 Starwell Maxi Nunc-Immuno Module Plates were coated with 150 μl of a 10 μg/ml solution of
a chicken affinity purified polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A). Plates were incubated
with the IgY overnight at 2-8°C and washed three times with 0.1M Glycine-HCl, pH 2.5. This wash was included to
liberate residual LF retained after the affinity purification of the antibody and was necessary to minimize the background. After 6 washes with PBS containing 0.05% TWEEN-20 (PBST), the anti-LF coated wells were exposed to
300 μl of a series of LF concentrations in neat plasma. The plates were incubated at 22°C for 2 hours. Plates were
then washed 6 times with PBST and 250 μl of 1.25 μM MAPKKide® Plus was added. The reaction was allowed to
proceed for 2, 3.5, and 5 hours at 37°C and overnight at ambient temperature. At each time point 200 μl of the
reaction mixture was removed from replicate wells and placed in HPLC sample vials.

HPLC was performed using a Zorbax Eclipse Plus C18 reverse phase column, 4.6 x 150 mm (Agilent) and a guard
column containing the same resin in a Varian ProStar HPLC system (Agilent). Solvent A was 0.1% TFA in water and
solvent B was 0.1% TFA in acetonitrile. The 16 minute HPLC method was as follows: 25% B for 0.75 minutes;
25 to 45% B in 4.75 minutes; 45 to 100% B in 0.75 minutes; 100% B for 3.75 minutes; 100 to 25%B in 0.67 minutes
and 5.34 minute equilibration with 25% B. The column effluent was monitored using an Hitachi fluorescence detector
with excitation set to 350 nm and emission at 450 nm to detect the free fluorophore cleaved from MAPKKide® Plus.
The injection volume was 20 µl. The 7-amido-4-methylcoumarin (AMC) peak retention time was 4.8 minutes.

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #769B – Anti-Lethal Factor from B. anthracis (Chicken lgY)
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor

…The MDS formulations were prepared with the following parameters: Matrix materials: poly (dl-lactide-co- glycolide) and poly (dl-lactide) Microsphere mean diameter range: 6-20 m RPA of anthrax (from List Biological Laboratories, Campbell, CA, US …

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

Serology:

…Toxin-neutralizing assay (TNA) was performed as published earlier [53] to qualitatively assess the ability of antibodies generated to neutralize lethal toxin. Briefly, heat inactivated individual sera were serially diluted in 8 log₂ steps with a starting dilution of 1:50 on sterile 96 well plates (BD Biosciences, Heidelberg, Germany) seeded with 10⁵ J774 A.1 macrophages per well. Lethal toxin (LF) was added at concentration of 500 ng/mL PA and 100 ng/mL LF (List Biological Laboratories, Campbell, CA, USA) and survival of macrophages was visualized through MTT (5 mg/mL, Alfa Aesar, Karsruhe, Germany), which is degraded by living cells to a measurable (OD540_nm) colour compound. …

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