Bacterial Toxin Research Citations

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

Reagents:

PA₆₃ was purchased from List Biological Laboratories (Campbell, CA). …

Channel reconstitution into planar lipid bilayers:

… For multichannel experiments, we applied 12 l of 0.2 mg ml¹ stock PA₆₃. Under this protocol, the channel insertions were always directional. The applied potential was defined as positive if it was higher on the side of protein addition.

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

TNA:

The titer of toxin-neutralizing antibody in serum was determined using modifications to the methods of Pitt et al [15] and Little et al [4] by testing the ability of the serum to inhibit the cytotoxicity of the combination of rPA with LF (List Biological Laboratories Inc., Campbell, CA, USA). The assay was carried by exposing J774A.1 murine macrophages (ATCC TIB-67) to PA and LF in the presence or absence of serum. In brief, J774A.1 cells were plated out on 96 well plates (SPL CO. Ltd, Pyeongtaek-si, Korea) and seeded with 10⁶ cells per well in 150 l volumes 2 hours before testing. …

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

Pre-incubation of MoDCs with toxins and chemicals:

MoDCs were incubated with PA (1 g/ml) and EF and/or LF (both from List Biological Laboratories Campbell, CA, USA) at 40 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
• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis

Toxins:

PA+FP59 was the toxin for cytotoxicity assays. … PA was also purchased from List Biological Laboratories (Campbell, CA).

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

Cell culture:

Bone marrow-derived macrophages (BMDM) were prepared by harvesting marrow from femurs and tibias of 129+/+ and Nlrp1b2/2 mice. Marrow was cultured for 7 d in IMDM containing 15% L cell-conditioned medium, 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). BMDM were incubated in OPTIMEM plus Glutamax (Life Technologies) containing varying concentrations of LT (List Biological Laboratories, Campbell, CA) for 4 h, and cell viability was assessed with the WST-1 reagent (F. HoffmannLa Roche, Basel, Switzerland). Peritoneal macrophages were elicited from wild-type, Nlrp1b2/2, Nlrp32/2, and Casp-12/2 mice with an i.p. injection of 4 ml 4% thioglycollate in PBS. Seventy-two hours after injection, the peritoneal cavity was lavaged with DMEM, and cells were plated and incubated for 1 h. Adherent cells were then washed twice with cold PBS and cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 150 U/ml penicillin, 150 mg/ml streptomycin, and 57.2 mM 2-ME (all from Life Technologies). For LT treatments, macrophages were primed overnight with 100 ng/ml ultrapure LPS (Invitrogen Life Technologies), washed with PBS, and incubated with OPTIMEM plus Glutamax containing either PBS or 1 mg/ml LT for 6 h. …

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