Citations

Reagents:

… The PA ligand was purchased from List Laboratories (Campbell, USA). …

Surface modification:

… After washing with HBS for 2 min at a flow rate of 30 m/min, 100 g/ml of PA was introduced and incubated for 30 min with a slow flow rate of 3 l/min for covalent binding to the SAM layer. …

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

Chemicals and Reagents:

PA and LF were purchased from List Biological Laboratories.

Toxin Treatment and Cell Viability Assays:

Mouse macrophages were treated with toxin for 4 h and human B lymphocytes for 48 h. Determination of macrophage viability was done by MTT assay as described in refs. 31, 32, whereas lymphocyte viability was determined by Resazurin (Invitrogen) fluorescence, as described by the manufacturer. Each data point shown in Figs. 1 and and22 represents the average and SD of results from three wells. Cell viability is shown as the percentage of survivors obtained relative to treatment by PA alone (100%). LD₅₀ was calculated for each cell line and normalized to account for variation in toxin potency from the two different lot numbers used, as determined in five and three independent LD₅₀ measurements of a control cell line for the first and second lots, respectively.

Immunofluorescence Microscopy:

PA protein was labeled with Alexa Fluor 647 using the protein labeling kit (A20173; Molecular Probes). We determined by MTT assay that such labeling did not affect the ability of PA/LF to kill macrophages. Fresh serum-free IMDM media was added to cells that contained 1 g/mL PA/Alexa Fluor 647. Cells were incubated for 20 min either at 4 C for PA-binding analysis or at 37 C for determination of PA internalization.

Biochemical Assay of PA Binding and Internalization:

Three million macrophage cells were exposed to 1 g/mL of PA at 4 C for 1 h for binding assay or at 37 C for 20 min for internalization assay. Cells were then washed with PBS solution three times and lysed in RIPA buffer containing a protease inhibitor mixture (Roche). Western blot analysis was performed using mouse monoclonal anti-PA antibody PA (C3) sc-52129 (Santa Cruz Biotechnology), or mouse antiactin monoclonal antibody (Sigma-Aldrich). Chemiluminescence of bands and their relative intensities were revealed using a VersaDoc 1000 instrument (Bio-Rad).

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 direct ELISA analysis of the peptide binding affinity to a recombinant PA protein, 83 kDa PA (PA 83 ; List Biological Laboratories, Campbell, CA, USA) was labeled directly with 44 kDa horseradish peroxidase enzyme (HRP) using EZ-Link plus activated peroxidase …

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

… Recombinant PA, EF, LF toxins and goat anti-PA primary antibody (Ab) were obtained from the List Biological Laboratories, Inc. … 

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

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

• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis
• Product #173B – Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis

… In a typical experiment, cells before stimulation were washed with serum-free medium, incubated for 1 h, and then stimulated in the conditions indicated in figure legends. LT, ET, protective antigen, lethal factor, and edema factor were purchased from List Biologicals. …

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).

PA-specific IgG ELISA:

Sera from individual rabbits were assayed in triplicate for the presence of PA-specific IgG by ELISA. Briefly, 96-well flat-bottomed Nunc Maxisorp plates (VWR International, Inc.) were coated with 0.1 g/well of PA (List Biological Laboratories, Inc.) overnight, blocked with 0.5% casein in phosphate-buffered saline (PBS) for 2 h at 37C, washed, and incubated for 1 h at 37C with the test serum. …

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

Materials and Methods – Anthrax Lethal Toxin:

Recombinant anthrax toxin PA and LF were obtained from List Biological Laboratories (Campbell, CA, USA). Antigens were snap frozen in liquid nitrogen and stored at 70 C in 10 L aliquots at a concentration of 1 mg/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

Materials:

Anthrax rPA and recombinant LF rLF were obtained from List Biological Laboratories, Inc. (Campbell, California). …

Macrophage Toxicity Assay

Mouse macrophages are sensitive to LeTx(the combination of PA and LF )-induced necrosis.. Here, rLF was added to the reconstituted powder formulation and the activity/stability of rPA was evaluated by measuring the PA dependent toxicity of rLF using mouse macrophage J774A.1 cells …

Anthrax lethal toxin neutralization assay:

Mouse macrophages are sensitive to LeTx(the combination of PA and LF )-induced necrosis. An in vitro lethal toxin (LeTx) neutralization assay using J774A.1 mouse macrophage cells was performed to determine the functional titer of the anti-PA antibody present in rabbit serum. The details of the assay were reported previously ³⁵. Briefly, rabbit sera were incubated with PA for 1 hr at 37C to allow neutralization to occur, followed by incubation with added LF at room temperature for 1 hr. The serum-PA/LF mixture was then added to the plate containing macrophage cells. The starting serum dilution on the final plate is 1:128 or 1:2⁷ with a final concentration of 0.1875 g of LeTx (rPA+rLF) per ml. Percent neutralization was calculated based on (sample OD value LeTx standard OD value)/(cells only OD value-LeTx standard OD value) 100. The percent neutralization was plotted vs serum log₂ dilutions and fitted with a second order polynomial curve. The serum dilution able to neutralize 50% of LeTx (NT50) was calculated. …

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

Immunization and infection of mice:

Mice were immunized by subcutaneous (s.c.) injection with 10 g of recombinant PA (List Biological Laboratories Inc.) and 0.3% aluminum hydroxide gel (Sigma-Aldrich) in 100 l of vaccine 20 days before infection, with a booster given 10 days before infection. …

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

Enzyme-Linked ImmunoSorbent Assay (ELISA):

96 well microtiter plates (EIR/RIA plate, Costar, Lowell, MA) were coated with 100 l rPA (List labs, Campbell, CA) at 1 g/ml in PBS overnight (o/n) at 4C. Plates were subsequently blocked with 200 l 1% BSA (in PBS containing 0.05% Tween 20, PBS-T) for two hours at 4C. …

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