Bacterial Toxin Research Citations

Reagents:

…The SEB and protective antigen (PA; List Biological Laboratories, Campbell, CA, USA) were fluorescently tagged using an amine reactive DyLight 488 …

MMS sorting:

In target sorting, the streptavidin-depleted library was resuspended in 1ml PBS and was incubated with 600nM SEB-biotin for 45min. After incubation, the cells were centrifuged at 3000g for 5min and resuspended in 1ml PBS containing 110⁹T1 streptavidin beads. After 45-min incubation with the magnetic beads, the cells were loaded onto the MMS and separated using the MMS positive selection program. The bacterial cells selected as SEB positive binders by the MMS were plated in serial dilutions to determine the resultant library diversity, and the remaining cells were grown overnight in LB-Cm²⁵. In the second, third, and fourth rounds of sorting, the SEB concentration was decreased from 600nM in round one to 300nM in round two, 150nM in round three, and 75nM in round four. …

Off-Cell affinity and specificity:

After blocking, a 45 min binding step was performed with each protein dissolved in PBST and serially diluted across each well in the 96-well plate: SEB-HRP initial concentration 14 nM, Strep-HRP initial concentration 250 nM, HRP initial concentration 200 nM, and PA-HRP initial concentration 100 nM. …

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

IPEC-J2 cell culture conditions and bacterial adherence assays:

…To test for the effects of endogenous LT expressed by the bacteria on adherence in isogenic porcine-origin ETEC strains that had not demonstrably shown an effect of LT on colonization [14], inocula were prepared and directly applied to the IPEC-J2 cells, as described above. To test the effect of exogenous LT on adherence on these strains, LT (List Biological Laboratories) at a concentration of 1, 10, or 100 ng/ml per well was added (pre-incubation) 1 h prior to bacterial inoculation. To test the potential of GM1 ganglioside to mitigate the effects of LT on adherence, LT at a concentration of 10 ng/ml or 100 ng/ml was incubated with the cells 1 h prior to their inoculation with bacteria, and 10 ng/ml or 100 ng/ml GM1 ganglioside (Sigma) was applied to the IPEC-J2 cells at the time of bacterial inoculation. Three independent experiments were performed for each assay.

Test of effects of different carbohydrates on LT production and secretion:

To test the effects of different sugars on LT secretion, CAYE media containing glucose, sucrose or fructose at 0%, 0.25%, 0.5%, 1%, or 2% were prepared. Strains were cultured in different media as described above, with aliquots taken at 0, 2, 4, and 6 h post-inoculation (PI). These samples were centrifuged (2500g, 10 min), and supernatants taken to analyze for LT concentrations by GM1-ELISA as previously described [17]. Three independent experiments were performed for each assay. 

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

Female A/J mice were vaccinated with rPA (1 mg; List Laboratories) formulated with Al(OH) 3 (200 m g), SMIP-7.10 (25 m g), or both.

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

Peptides and proteins:

… Recombinant protective antigen (rPA) was obtained from List Biologicals (Campbell, CA ) ….

 Vaccines and immunization:

… For anthrax and influenza protein vaccines used in Chapter 3, lipids were prepared with S100 lecithin (Lipoid GmBH, Bermany).  These vaccines were delivered via intramuscular injections of 25 L on each the right and left leg.  Each 50 L dose of anthrax vaccine contained 1 g rPA and adjuvants as indicated in figure legends.  …

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

…MAPKKide was purchased from List Biological Laboratories (Campbell, …

Author did not specify which MAPKKide was utilized.  List Labs provides the following:

• Product #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor
• Product #531 – MAPKKide Peptide Substrate (DABCYL/FITC) for Anthrax Lethal Factor

TNA Assay:

The TNA Assay measures the levels of anthrax lethal toxin neutralizing antibody using an in vitro cytotoxicity assay. The assay was originally validated at the CDC, but was then transferred and validated at Battelle, where the testing of these serum samples occurred[8, 9]. Briefly, microtiter cell plates were seeded with J774A.1 cells and allowed to adhere. In separate microplates a mixture of recombinant protective antigen (rPA, List Biological Laboratories, Inc., Campbell, California, Cat. No. 171B) and recombinant lethal factor (rLF, List Biological Laboratories, Inc., Campbell, California Cat. No. 172B) was added to serial dilutions of the test samples and controls and incubated prior to transfer to the cell plate. The final concentration of rPA was 0.05 g/mL and the final concentration of rLF was 0.04 g/mL. MTT was then added to the cell plates to allow viable cells to reduce the MTT dye. …

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis

Methods:

Recombinant LF (native sequence) was purchased from List Biological Laboratories, Inc. (Campbell, CA), and used without further purification. …

LF peptidolytic activity assay:

Cleavage of the synthetic MAPKK substrate by LF results in separation of the donor and acceptor moieties and restoration of fluorescence. Development of fluorescence as read by a SpectraMax M2 multiwell microplate fluorometer (Ex/Em=490nm/523nm) was used to measure the rate of LF-catalyzed substrate cleavage Kocer SS, Walker SG, Zerler B, et al. Metalloproteinase inhibitors, nonantimicrobial chemically modified tetracyclines, and ilomastat block Bacillus anthracis lethal factor activity in viable cells. Infect Immun 2005;73:754857 [Google Scholar]. All reagents were diluted as necessary for each assay just prior to use. Assays were repeated until conditions were optimized and consistent results were achieved. Reactions for data collection were then carried out in triplicate, in 96-well microplates, at 37C, with a final volume of 150L in each well: 50L of LF, 50L of substrate and 50L of inhibitor (or reaction buffer for control wells). LF was diluted with reaction buffer to a 100nM final concentration in the well under all reaction conditions, while inhibitor and substrate were diluted to various concentrations, in reaction buffer, as described in the results. All LF and inhibitor/control pairs were incubated at 37C in the reaction wells for 20min prior to the addition of substrate, and read by the fluorometer immediately after the addition of substrate. …

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).

Anthrax protective antigen and hepatitis B surface antigen ELISAs:

Ninety-six-well microtiter plates (Corning, Lowell, MA) were coated with 1 g/well of recombinant PA (List Biological Laboratories, Campbell, CA) …

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

Memory B Cell ELISPOT:

… Cultured PBMCs were washed then plated at 1:2 serial dilutions (3.75 10⁵ 1.6 10⁴ cells/well) in triplicate onto ELISPOT plates coated with 10 g/mL PA (List Biologicals, Campbell, CA, USA) … The frequency of PA-specific ASCs was defined as: [(anti-PA IgG spots per 100,000 input cells)/(total IgG spots per 100,000 input cells)] 100.

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

Reagents:  the receptorbinding protein protective antigen and metalloprotease lethal factor were from List Biological

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