Bacterial Toxin Research Citations

… 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

Vaccines and immunization:

…Anthrax vaccine was prepared with recombinant protective antigen (rPA; List Biologicals, Campbell, CA) to deliver 1 g antigen, 1 g poly I:C, and/ or 1 g Pam3CSK4 per 50 L dose.

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

Antibody assessment:

Recombinant proteins (rPA and rLF) were …or purchased from List Biological Laboratories (Campbell, CA). Antibody titers against PA were determined by enzyme-linked immunosorbent assay (ELISA). Plates were coated with rPA at 2.0ng/L in a volume of 50L/well and incubated overnight at 4C. Plates were then washed three times with 1wash solution (KPL, Gaithersburg, MD), and blocked with 1% bovine serum albumin (KPL) containing 1% non-fat dry milk for 1h at 37C. …

The lethal toxin-neutralizing assay (TNA) titer was determined as described³⁴ except J774A.1 murine macrophage cells were used. Briefly, the serially diluted sera were pre-incubated with toxin mixture (PA and LF at 5:4 ratio) at 37C for 30min before adding to J774A.1 cells in 96-well plates. …

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 [23]. Briefly, 96-well plates were coated with 1 g/well of recombinant PA, LF (List Biologicals, 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

Macrophage culture:

…BMDMs were primed for 4h with 600ngml¹ E. coli 0111:B4 LPS… and then transfected with poly(dA:dT)…, lethal toxin (500ngml¹, #172D and #171D mixed at a 50/50 ratio, List Labs) or flagellin…

• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Proteins:

…Protective antigen (PA) from Bacillus anthracis (List Biological Labs, Campbell, CA, USA) was used in the assay at a final concentration of 3 g/ml. …

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