Bacterial Toxin Research Citations

… 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

In vitro characterization of plant-produced mAbs:

ELISA was performed as previously described in reference 36, with slight modifications. Plates were coated with 1 g/mL of recombinant B. anthracis PA (List Biological Laboratories Inc., Campbell, CA). …

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

Protein Purification:

Highly purified recombinant proteins were prepared in our laboratories for binding assays as well as immunizations, as described below. For ELISA and LTx neutralization assays, purified PA and LF were purchased from List Biologicals Laboratories, Inc., 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

LF quantification:

… To achieve the necessary dynamic range and to account for high-sensitivity rapid-response sample analysis, two standard curves were prepared, one for 20 ng/ml, l (low volume [LV]) and the other for 200 ng/ml, l (high volume [HV]) serum volumes. In addition, three quality control samples (QCs) were prepared in human serum pools (Interstate Blood Bank, Memphis, TN) using recombinant LF (List Biological Laboratory, Campbell, CA) and validated. …

PA antigen capture immunoassay:

Microtiter plates (Thermo Labsystems, Franklin, MA) were coated with monoclonal anti-PA antibody AVR1046 obtained by the same methodologies used for anti-LF monoclonal described previously (4) at 1.28 g/ml in 0.01 M phosphate-buffered saline (PBS), pH 7.4. Coated plates were washed with a wash buffer consisting of PBS and 0.1% Tween 20, pH 7.4. Recombinant PA (rPA; List Biological Laboratories, Inc., Campbell, CA) was serially diluted twofold using a 1/25 or a 1/100 dilution of normal rhesus macaque serum (RMS). The rPA dilutions formed a seven-point standard curve from 0.6 to 37.5 ng/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