Bacterial Toxin Research Citations

Materials: 

… PA toxin was purchased from List Biological Laboratories, Inc. (Campbell, CA, USA). …

Cloning:

In the ACE process the aptamers were initially labeled with a 6-carboxyfluorescein and incubated with increasing concentration of PA Toxin. …

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

Recombinant Protein ELISAs:

Ninety-six well plates were coated overnight at 4 C with 1 g/well of rLF or rPA (List Biologicals, Campbell, CA). After washing with PBS-Tween and blocking with PBS/BSA, diluted pre-immune and Day 28 (4 days after a second booster) serum from each mouse was added in duplicate and incubated for 2 h at room temperature (RT). …

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

Challenge with anthrax toxin or MEK pathway inhibitors:

At indicated time points, cultured cells were treated with fresh medium containing 2 g/ml PA or 2 g/ml LT (2 g/ml PA plus 2 g/ml LF), both from List Biological Laboratories, Inc., Campbell, CA. An equal volume of diluent was used as a negative control. Duration of treatment was 24, 72, or 144 h or as indicated. …

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

Measurement of cytotoxicity of diphtheria and anthrax toxin:

… To determine the protective effect of the purified nanobodies on anthrax toxin cytotoxicity, RAW cells were pre-incubated with 20 M purified nanobodies or 10 M D9R in 48-well plates (Costar) for 30 min at 37 C. Subsequently, 200 ng/ml PA (protective antigen; List Biological Labs) and 400 ng/ml LF (lethal factor; List Biological Labs) were added and the cells were incubated for 1.5 h, with one medium change after 45 min. The cell viability was determined using the MTT assay described above.

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

Expression and Labeling of M18 scFv :

A standard 3-ml culture of cells harboring plasmids to surface display M18 scFv (e.g. pBAD_M18_138) was grown in LB medium (BD Diagnostics) in the presence of 30 g/ml chloramphenicol (Thermo Fisher Scientific) to an optical density (A₆₀₀) of 0.6 at 25 C. Cells were then induced via the addition of 0.2% l-arabinose (Sigma) to express M18 scFv for 12 h at 25 C. The presence of M18 scFv on the surface of E. coli was characterized by the ability of intact cells to bind antigens (PA₆₃ and PAD4) using flow cytometry. PAD4 containing FLAG epitope tag (rPAD4) was recombinantly expressed in E. coli and purified as described previously (35), and PA₆₃-FITC was obtained from List Biological Laboratories, Inc. …  

• Product #175 – Anthrax PA 63 – FITC Conjugate                               

For Western blots, whole-cell lysates from 106 J774A.1 cells, separated by SDS/PAGE, were probed by anti-PA (List Biological Laboratories) and anti-actin antibodies (Santa Cruz Biotechnology) with chemiluminescence detection. …

 Materials:

The dominant negative inhibitor antigen was produced by Pharmaceutical Solutions LLC
(Bloomington, IN) as a lyophilized powder in sealed glass vials. The vials of the DNI protein
contained 25 mg of protein, 113 mg mannitol, 33 mg sucrose, and 2.4 mg dibasic sodium
phosphate. They were reconstituted with 3.3 ml of water for injection (WFI), the
reconstituted protein concentration was 91 M . 10 mg of recombinant protective antigen was
also purchased from List Biological Laboratories Inc (Campbell, CA). …

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

Materials:

… Nicked anthrax PA 63 from B. anthracis was obtained from List Biological Laboratories Inc., Campbell, CA. …

Binding Experiments:

The binding of the His-tagged proteins to the C2II-channel and the binding component PA₆₃ was investigated with titration experiments similar to those performed previously to study the binding of 4-aminoquinolones to the C2II- and PA₆₃-channels and EF and LF to the PA₆₃-channel in single- or multi-channel experiments [38][40]. The aqueous phase contained always 150 mM KCl, buffered with 10 mM MES-KOH to pH 5.5 to pH 6. The C2II- and PA₆₃-channels were reconstituted into lipid bilayers. About 60 minutes after the addition of either activated C2II or PA₆₃ to the cis-side of the membrane, the rate of channel insertion in the membranes was very small. Then concentrated solutions of His-tagged proteins were added to the cis-side of the membranes while stirring to allow equilibration.

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

Vaccine formulations:

The vaccines discussed in this report were composed of rPA (List Biological Laboratories), recombinant spore-specific proteins produced as described earlier ( Brahmbhatt et al., 2007 ; Cybulski et al., 2008 ; Mikesell et al., 1983 ) or a combination of the antigens. The specific amounts of antigens used in the vaccine formulations are described in the figure legend of each experiment. Aluminium hydroxide gel (AL; aluminium content per vaccination ~125 g for mice or ~500 g for guinea pigs) or the Sigma adjuvant system (SAS) was used as adjuvant (Sigma Aldrich). SAS is an emulsion of monophosphoryl lipid A and trehalose dicorynomycolate, which may promote mixed T helper 1 (Th1)/Th2 immune responses to vaccines, and was used as directed by the manufacturer.

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

Experimental Protocols:

Rats received intravenous (femoral vein) LeTx [lethal factor (LF) and protective antigen (PA); LF 100 g/kg+PA 200 g/kg; infusion rate 1.0 ml/hr) or vehicle (PA+PBS with 1% BSA, 0.5 ml/hr or PBS with 1% BSA, 1.0 ml/hr]. LF and PA were recombinant proteins prepared from B. anthracis and purchased from List Biological Laboratories. Experiments were completed in INT and SAD rats. MAP, HR, lumbar SND, and renal SND were recorded continuously during LeTx or vehicle infusions. The maximum infusion duration was 3 hours; however, due to LeTx-induced reductions in MAP, the majority of experiments were terminated before the completion of 3 hours. The effect of a short bout of combined hypoxia and hypercapnia on SND responsivity, produced by shutting off the mechanical ventilator for a short period of time (1530 s), was determined near the end of LeTx and vehicle infusion experiments. Arterial pO2 and pCO2 levels were measured before and 1530 s after ventilation was terminated. Rats were euthanized by an intravenous overdose of methohexital sodium (150 mg/kg iv).

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