Bacterial Toxin Research Citations

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

We next tested the ability of RDI to neutralize LeTx containing an antibody-resistant form of PA (PA^L685A) in a rat model system. Male Harlan Sprague-Dawley rats (180 to 200 g; Harlan, Indianapolis, IN) were anesthetized with isofluoranes and inoculated with 500 l LeTx mixture by intravenous injection in the tail vein. The anthrax LeTx was prepared for each group by mixing 12 g LF (List Biological Laboratories, Campbell, CA) with 40 g PA (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).

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

For the procurement of positive control hyperimmune anti-PA and anti-LF serum for use in the western blotting and as positive controls for the LeTx neutralization assays, rabbits were immunized with 250 g of either soluble PA83 or LF in an emulsion with CFA (List Biological Laboratories, Campbell, CA). Rabbits were then boosted three times at 23-week intervals with 125 g of the respective immunogens in an emulsion with incomplete Freund’s adjuvant …

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

Samples of LF used for comparison were purchased from List Biological Laboratories (Campbell, CA).

Results – Toxicities of LF proteins produced from avirulent B. anthracis strains:

… B. anthracis strains cured of the pXO1 and pXO2 virulence plasmids [18]. LF expressed from pSJ115 is here termed LF-HMA to denote the presence of the two residues His-Met (HM) added at its N-terminus due to the cloning manipulations. This expression system is licensed to List Biological Laboratories (Campbell, CA), and the LF sold by them is therefore also LF-HMA. …

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

Microinjection of Toxin Proteins and Use of Inhibitors:

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

Quantification of PA₈₃ bound to chimeras 206 and 264:

Recombinant PA₈₃ (List Biological Laboratories) in 5 mM Hepes, 50 mM NaCl (pH 7.5) was mixed with purified chimeras 206 and 264 in 50 mM Hepes (pH 7.5) in a ratio of 180:1 (equimolar amounts of PA₈₃and VWA domains). Following incubation for 20 min at room temprature, an aliquot from each of the samples was removed and stored at 20 C pending analysis. The remainder of the samples was transferred to an ultracentrifuge tube and underlayed with a 30% (w/w) sucrose cushion in 50 mM Hepes (pH 7.5). Complexes of chimeras decorated with PA₈₃ were pelleted by centrifugation at 200,000g for 45 min. …

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