Bacterial Toxin Research Citations

TNA Assay:

The TNA Assay measures the levels of anthrax lethal toxin neutralizing antibody using an in vitro cytotoxicity assay. The assay was originally validated at the CDC, but was then transferred and validated at Battelle, where the testing of these serum samples occurred[8, 9]. Briefly, microtiter cell plates were seeded with J774A.1 cells and allowed to adhere. In separate microplates a mixture of recombinant protective antigen (rPA, List Biological Laboratories, Inc., Campbell, California, Cat. No. 171B) and recombinant lethal factor (rLF, List Biological Laboratories, Inc., Campbell, California Cat. No. 172B) was added to serial dilutions of the test samples and controls and incubated prior to transfer to the cell plate. The final concentration of rPA was 0.05 g/mL and the final concentration of rLF was 0.04 g/mL. MTT was then added to the cell plates to allow viable cells to reduce the MTT dye. …

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

Methods:

Recombinant LF (native sequence) was purchased from List Biological Laboratories, Inc. (Campbell, CA), and used without further purification. …

LF peptidolytic activity assay:

Cleavage of the synthetic MAPKK substrate by LF results in separation of the donor and acceptor moieties and restoration of fluorescence. Development of fluorescence as read by a SpectraMax M2 multiwell microplate fluorometer (Ex/Em=490nm/523nm) was used to measure the rate of LF-catalyzed substrate cleavage Kocer SS, Walker SG, Zerler B, et al. Metalloproteinase inhibitors, nonantimicrobial chemically modified tetracyclines, and ilomastat block Bacillus anthracis lethal factor activity in viable cells. Infect Immun 2005;73:754857 [Google Scholar]. All reagents were diluted as necessary for each assay just prior to use. Assays were repeated until conditions were optimized and consistent results were achieved. Reactions for data collection were then carried out in triplicate, in 96-well microplates, at 37C, with a final volume of 150L in each well: 50L of LF, 50L of substrate and 50L of inhibitor (or reaction buffer for control wells). LF was diluted with reaction buffer to a 100nM final concentration in the well under all reaction conditions, while inhibitor and substrate were diluted to various concentrations, in reaction buffer, as described in the results. All LF and inhibitor/control pairs were incubated at 37C in the reaction wells for 20min prior to the addition of substrate, and read by the fluorometer immediately after the addition of substrate. …

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 #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

Anthrax protective antigen and hepatitis B surface antigen ELISAs:

Ninety-six-well microtiter plates (Corning, Lowell, MA) were coated with 1 g/well of recombinant PA (List Biological Laboratories, Campbell, CA) …

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

Memory B Cell ELISPOT:

… Cultured PBMCs were washed then plated at 1:2 serial dilutions (3.75 10⁵ 1.6 10⁴ cells/well) in triplicate onto ELISPOT plates coated with 10 g/mL PA (List Biologicals, Campbell, CA, USA) … The frequency of PA-specific ASCs was defined as: [(anti-PA IgG spots per 100,000 input cells)/(total IgG spots per 100,000 input cells)] 100.

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

Reagents:  the receptorbinding protein protective antigen and metalloprotease lethal factor were from List Biological

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

… and PA was obtained from List Biological Laboratories (Campbell, CA, USA). …

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

Reagents:

For the bilayer lipid measurements, PA 63 was purchased from List Biological Laboratories, Inc. (Campbell, CA, USA).

Channel Reconstitution into Planar Lipid Bilayers:

…Single channels were formed by adding 0.5 to 1 L of 20g mL 1 solution of PA63, or 0.2 to 0.5 L of 48 ng mL 1 solution of C2IIa to the 1.5 mL aqueous phase on the cis-half of the bilayer chamber. For multichannel experiments, we applied 12 L of 0.2 mg mL1 stock PA63 or 12 L of 48g mL 1 stock C2IIa to the cis-side of the membrane. Under this protocol, PA63 and C2IIa channel insertions were always
directional as judged by channel conductance asymmetry in the applied transmembrane voltage…In most of the experiments, the PAMAM dendrimers were added to the cis-compartment of a bilayer chamber, which was the side of PA63 and C2IIa addition.

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

General reagents:

…Anthrax PA83 was purchased from List Biological Laboratories (Campbell, CA, USA). …

MPII proteolysis of proteins in vitro:

Anthrax PA83 (2 g; ~ 1 m) and the following purified human proteins the furin 27107 prodomain (1.8 g, ~ 9 m) [20], the MT1MMP proenzyme (2 g; ~ 2 m), MBP (2 g; ~ 5 m) and BTN3A2 (2 g; ~ 2.5 m) were each coincubated for 1 h at 37 C with increasing concentrations of MPII in 50 mm Hepes, pH 8.0, containing 1 mm CaCl₂, 0.5 mm MgCl₂ and 10 m ZnCl₂. As a control, PA83 and the MT1MMP proenzyme were coincubated for 1 h at 37 C with furin (at the 1 : 100 to 1 : 10 000 enzyme/substrate molar ratio) in 20-L reactions containing 100 mm Hepes, pH 7.5 and 1 mm CaCl₂. …

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

Materials:

… Activated protective antigen [63 kDa (1 M=63g/ml); PA63] and recombinant lethal factor [90 kDa (1 M=90 g/ml); LF] were obtained from List Biological Laboratories (Campbell, CA). …

PA63 and LF were used in the following:

• Optimization of LTx complex detection assay
• Limit of detection and quantification
• Precision and Accuracy for PA83 and PA63 Detection
• Detection of PA63 and LTx in the presence of AIG

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

Cytotoxicity assay:

PA and LF were purchased from List Biological Laboratories, Inc., …

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