Bacterial Toxin Research Citations

… Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native, List Biological Laboratories, Cat#169A. Anthrax Protective Antigen (PA), Recombinant from B. anthracis, List Biological Laboratories, Cat#171E. …

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

List Labs Product #169A (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Lyophilized) has been discontinued; however, Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Liquid) is available for purchase.

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

Briefly, heat-extracted CFA/I, 389 CS1, CS2, CS3, CS4 or CS5 fimbriae (200 ng per well) and LT (List Biological Laboratories, Inc., Campbell, CA; 100 ng per well) were coated to wells of 2HB plates …

Lethal Toxin (LT) consists of LF + PA:

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

Quantification of anti-PA IgG antibodies:

A 96-well plate (Immulon 2 HB Southern Biological, Birmingham, AL, USA) was coated with 100 l of rPA coating solution (1 g/ml in PBS, List Biological Laboratories, Inc. Campbell, CA, USA). …

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

Chemicals, reagents, equipment and safety:

… Recombinant LF (rLF) was purchased from List Biological Laboratories (Campbell, CA).

Sample preparation:

The MS method for anthrax LF relies on three simple steps that each add a layer of sensitivity and specificity (Fig. 1). LF-MABs selectively purify and concentrate total-LF (LF + LTx) (Step 1). Immobilized LF is reacted with an optimized peptide substrate which it hydrolyzes producing two unique LF-specific product peptides (Step 2). Because each LF molecule can continuously hydrolyze peptide substrate, this step allows amplification of the resulting signal and improves detection. Analysis of the cleaved peptides by high-sensitivity/high-resolution isotope-dilution (ID) MALDI-TOF MS (Step 3). This strategy has been successfully applied to several enzymatic protein toxins [22].

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).

Antigens:

… Recombinant protective antigen (rPA) was obtained from List Biological Laboratories (Campbell, CA). Each vial was reconstituted  

Quantification of anti-PA IgG antibodies:

Wells of an Immulon 2 HB, 96-well round bottom microtiter plate (Southern Biological, Birmingham, AL) were coated with 100 l of a coating solution (rPA diluted at 1 g/ml in a 0.01 M PBS solution, pH 7.4). …

Titration Curve:

Eight rPA-coated microtiter plates were prepared. Following overnight incubation, approximately 200 l of REF plasma that had been diluted to 1:500 was added to the first well of three rows on each plate then two-fold serial dilutions carried out. …

Specificity:

In the second experiment, two rPA coated microtiter plates were prepared, except that ovalbumin (100 l diluted at 1 g/ml in a 0.01 M PBS solution), a heterologous protein to B. anthracis PA, was also used to coat six wells in each plate. …

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

… Briefly, 10 ng STa-ovalbumin conjugates (provided by Dr. DC Robertson at Kansas State University) was used to coat each well of Costar plates (Corning Inc., Corning, NY) to titrate anti-STa antibodies, and 100 ng LT (List Biological Laboratories, Inc., Campbell, CA) was coated to each well of 2HB plates (Thermo Scientific, Rochester, NY) to titrate anti-LT antibodies. …

Lethal Toxin (LT) is PA + LF:

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

Lethal toxin and signaling inhibitors:

Recombinant protective antigen and wild-type lethal factor or lethal factor mutant (E687C) were obtained from List Biologicals (Campbell, CA) …

In vitro lethal toxin assay with Rag1^-/- splenocytes or human tonsillar lymphocytes:

100,000500,000 splenocytes per well were incubated in a round bottom 96 well plate in IMDM media supplemented with 10% FBS, 100 U/ml pencillin, 100 U/ml streptomycin, IL-2 (20 ng/ml) and IL-7 (10 ng/ml). Human tonsillar lymphocytes (2×10⁶) were plated in 200 l RPMI media supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, non-essential amino acids, sodium pyruvate, IL-2 (20 ng/ml), IL-7 (10 ng/ml) and IL-1 (20 ng/ml). For initial lethal toxin experiments splenocytes/tonsillar lymphocytes were cultured in media containing in 0.1% serum to minimize the effect of serum on lethal factor enzyme activity. After the initial period of lethal toxin treatment (3 hrs), serum was replenished to a full concentration of 10%. Cells after toxin treatment were stimulated with recombinant mouse or human IL-23 (50 ng/ml) (eBioscience) for 18 hrs. Cell supernatants were harvested by centrifugation at 1,500 rpm for 5 minutes and then used for the measurement of IL-22.

In vitro lethal toxin assay with MNK-3 cells:

MNK-3 cells (an ILC3 cell line) were cultured in MNK-3 media (DMEM (High glucose), 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM Glutagro, 1 mM sodium pyruvate, 10 mM HEPES, 55 M -mercaptoethanol, 5 g/ml gentamicin, 10 ng/ml IL-7 and 10 ng/ml IL-15). For lethal toxin experiments MNK-3 cells were cultured in media containing 10 ng/ml IL-7. Cells were treated with lethal toxin for 2 hrs followed by IL-23 stimulation for 6 hrs. Cell lysates were analyzed for RNA and supernatants were analyzed for secreted IL-22 by ELISA.

Lethal Toxin (LT) = PA + LF

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

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

… Given the important role of pH change in the anthrax toxin internalization process, the current study is designed to address the disparity.

Material and Methods:

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

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

Cells and Reagents:

… Lyophilized recombinant PA and LF were purchased from List Biological Laboratories, Inc. (Campbell, CA), and reconstituted in sterile water to make stock solutions with final concentrations of 1 mg/mL. …

Cell Proliferation Analysis:

Eighty thousand Hepa1c1c7 cells were added into individual wells of 12-well plates. Six replicates were used for each group. The cells were treated with or without LT for 48 h. The cell number in each well was counted after the treatment using a TC20 automated cell counter (BioRad, Hercules, CA). LT-induced cell proliferation inhibition was calculated using the following formula: …

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

Toxin neutralization assay (TNA):

An in vitro toxin neutralization assay (TNA) was performed using a MTT [3-(4.5-dimethylthiazol-2-yl)2.5-diphenyltetrazolium bromide] in a colorimetric cell viability assay with the J774A.1 macrophage cell line as previously described by Hering et al. [18] with slight modifications by Ndumnego et al. [17]. Briefly, serial diluted goat serum was incubated with PA and LF (List Biological Laboratories, USA) at concentrations of 500 and 400 ng/mL respectively before addition to overnight incubated macrophage cells. …

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