Bacterial Toxin Research Citations

Materials and Reagents:

Anthrax lethal toxins (PA and LF) and Escherichia coli O111:B4 LPS were purchased from the List Biological Laboratories.

Author did not specify which List Labs Lethal Factor was utilized.  List Labs offers Lethal Factor Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

RESULTS:

HDAC8 inhibition restores the production of pro-IL-1 in response to LPS in LeTx-exposed murine macrophages. LeTx suppresses expression of various inflammatory cytokines through inactivating MEKs (3-5). Consistent with these reports, a sub-lethal dose of LeTx (100 ng/ml PA & LF, a.a. for 4 h) completely cleaved MEK1 and inhibited expression of pro-IL-1 induced by LPS (100 ng/ml) for more than 2 days in RAW264.7 macrophages (Fig. 1A). …

… Alternatively, macrophages were stimulated using E. coli lipopolysaccharide (100 ng ml 1 ; Sigma-Aldrich, St Louis, MO, USA), Anthrax protective antigen and Anthrax lethal factor (List Biological Laboratories, Campbell, CA, USA), …

• Product #171 – Anthrax Protective Antigen (PA),Recombinant from B. anthracis
• Author did not specify which List Labs Lethal Factor was utilized.  List Labs offers Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

ELISPOT:

Peripheral blood mononuclear cells (PBMCs) were prepared and washed in RPMI medium supplemented with 10% fetal calf serum, penicillin, streptomycin, and L-glutamine. …The antigen, recombinant Protective Antigen (rPA), LF or EF (List Biological Laboratories Campbell, Calif.) was added at a final concentration of 12.5 g/ml for PA or 25 g/ml for either LF or EF. The plates were incubated overnight at 37C in a 5% CO₂, humidified atmosphere. …

Quantitative anti-PA IgG ELISA:

Wells of 96-well microtiter plates (Immulon 2HB; Dynex Technologies, Chantilly, Va.) were coated by adding of 100 l rPA (List Biological Laboratories Campbell, Calif.) diluted to 1 g/ml in PBS. Plates were incubated overnight at 4C. …

Lethal toxin neutralization assay (TNA):

…Lethal toxin (LeTx; 100 ng of rPA/ml and 50 ng of LF/ml, final concentrations) was prepared in the same medium and pre-incubated with the sample dilutions in a humidified incubator set at 37C, 5% CO2 for 1 h. LeTx was prepared using PA obtained from List Biological Laboratories …

End-point ELISA for LF and EF:

Wells of 96-well microtitre plates (Immulon 2HB) were coated with either LF or EF (List Biological Laboratories) at 1 g/ mL in PBS (100 L per well). …

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

 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).

Chemicals and Reagents:

… Recombinant anthrax lethal factor (LF) from B. anthracis was from List Biological Laboratories (Campbell, California).

Characterisation of the thresholds for LF and EF detection:

In a first step, we characterised the threshold for the enzymatic quantification of LF and EF by using plasma and ear tissue samples derived from mice infected…

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).

Bacterial Strains and Reagents:

… Activated protective antigen was procured form List Biological Laboratories (USA). …

2.6. Binding to LF and Native-PAGE

For the confirmation of functional activity of the expressed PA, 2g of trypsin digested PA was incubated with 2g of LF at room temperature for 30 minutes. The samples were run on 4% native polyacrylamide gel at 4C and visualized by staining the gel with coomassie brilliant blue.

2.7. Animal Immunization

Six female Balb/c mice were procured from Institutional Animal Care Center, DRDE, and were provided with food and water ad libitum. Each mouse was immunized subcutaneously with 10g PA adsorbed on aluminium hydroxide adjuvant on days 0, 14, and 28. Mice receiving only PBS were taken as control. All mice were bled prior to first immunization on day 0 and on 35th day. Serum was separated and stored at 20C until further use.

2.8. Enzyme Linked Immunosorbent Assay

To test the reactivity of PA immunized sera, indirect ELISA was performed by coating 1gmL¹ PA in ELISA plate overnight at 4C. After blocking with 5% skim milk for one hour, serial twofold dilution of PA immunized sera starting from 1:1000 was added and incubated for one hour at room temperature. …

2.9. Cell Culture

Mouse macrophage cell line J774.1 was procured from National centre for cell sciences, Pune, India and was maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100UmL¹ penicillin and streptomycin as antibiotics. One day before the experiment 4 10⁴ cells were seeded in a 96 well plate and incubated at 37C in presence of 5% humidified CO₂. The next day spent media was replaced by a fresh medium containing gradient concentration of soluble PA and native PA of B. anthracis from List Biologicals (0.005gmL¹ to 5gmL¹) and was incubated at 37C along with 0.125gmL¹ LF. After 4 hours, 20L of 5mgmL¹ MTT (3-(4,5 dimethylthiazol-2 yl)-2,5 diphenyl tetrazolium bromide) was added to each well and further incubated for 30 minutes.

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) 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.…

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°Chourly 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

… The light chain of botulinum neurotoxin type B (50 kDa, List Biological Laboratories, Campbell, CA) was used to cleave recombinant Sb2.13 …

• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant

Toxin Neutralization Assay:

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

Anthrax lethal toxin neutralization assay:

Samples collected after release were characterized for toxin-binding activity via an anthrax LeTx neutralization assay that was performed using decreasing amounts of the released samples. Specifically, the biological activity of PANG was measured using a LeTx neutralization assay as described previously with modifications.³⁵ J774A.1 (ATCC TIB-67, Manassas, VA) cells were plated in 96-well flat-bottomed tissue culture plates at 2.5 10⁴ cells/well in 50 L and incubated for 1619 h at 37C with 5% CO₂. Test samples were diluted and titrated on the plated cells in the presence or absence of LeTx (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).

Analysis of TMV-PA Antigens for Reactivity against PA Specific Antibodies:

SDS-PAGE, ELISA and Western Blots – 
 
… For westerns, gels were transferred onto 0.2 um nitrocellulose
membranes (7 cm x 8.5 cm) in Tris-Glycine buffer (25 mM Tris, 192 mM Glycine, 20%
Methanol, pH 8.3) for 1 hour at 100V. Membranes were blocked with 2.5% non-fat milk
in TBS (20 mM Tris, 150 mM NaCl, pH 7.6) for 1 hour at room temperature. Primary
antibody (Goat anti-PA polyclonal, List Biologics Lot # 7712A2 …

Cell culture and reagents:

… Protective antigen (PA), lethal factor (LF) and Escherichia coli derived LPS were purchased from List Biological Laboratories Inc. (California USA). The following is a list of antibodies (Abs) used in this study. …

Long-term TIR cells were generated as previously described [110].  Briefly, RAW 264.7 macrophages were treated with a cytolytic dose of LeTx (250 ng/mL LF and 500 ng/mL PA) for 5 h and surviving cells were plated in fresh culture medium.  After two weeks, surviving clones were individually picked and plated on a 96-well plate.  Each clone was tested for LeTx sensitivity and resistant clones were pooled and propagated. Short-term TIR cells were generated as previously described [110].  Briefly, RAW 264.7 macrophages were treated with a sub-lethal dose of LeTx (100 ng/mL LF and 100 ng/mL PA) for 5 h and then supplemented with fresh media overnight.  The next day surviving cells were pooled and plated onto new culture plates with fresh media.  BMDM-TIR cells were generated by Dr. Soon-Duck Ha as previously described [177].  Briefly, BMDMs were treated with a sub-lethal dose of LeTx (100 ng/mL LF and 100 ng/mL PA) for 24 h and surviving cells were plated onto new culture dishes with fresh media. …

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