Bacterial Toxin Research Citations

Intestinal organoid culture and live imaging:

Primary intestinal epithelial organoids were grown as described before⁵⁶. Briefly, the small intestine and colon were flushed and cut into small pieces that were dissociated in PBS containing 2mM EDTA for 30min at 4C. After extensive washing, the isolated crypts were pelleted and mixed with 25l of Matrigel (Corning) and put in a 24-well plate. After polymerization of the Matrigel, complete culture medium containing advanced DMEM/F12 (Gibco) supplemented with B27 supplement (0.02%, Invitrogen), N2 supplement (0.1%, Invitrogen), N-acetylcysteine (0.0025%, Sigma-Aldrich), mouse epidermal growth factor (mEGF; 0.001%, Invitrogen), and conditioned Rspondin and mNoggin medium was added to the wells. Organoids were seeded and imaged in an 8-well chamber (iBidi) for cell death analysis by real time lapse microscopy or in 24-well plates for Western blotting analysis. Cell death was induced with TcdA (1g/ml, Enzo Life Sciences), TcdB (1g/ml, List Laboratories), …

• Product #155 – Toxin B from Clostridium difficile

Toxin preparation:

C. difficile toxin was prepared as previously described (87). Briefly, C. difficile strain r20291 (AB and AB⁺) was cultured in tryptone-yeast extract media for 24 hours in an anaerobic chamber at 37C. Cultures were diluted to an OD that corresponds to 2 10⁷ CFU/ml and spun down; the supernatant was sterilized using 0.22 M filters. Supernatant prepared from toxin-negative (control) and toxin-positive strain was used to stimulate BMDMs at a 1:5 dilution.

Purified TcdB was obtained from List Biological Laboratories (catalog 155) and used at final concentration of 0.5 g/ml, as previously described (7).

• Product #155 – Toxin B from Clostridium difficile

… Bordetella pertussis Adenylate Cyclase Toxin/toxoid (Act) was purchased from Sigma (Cat#A0847, Sigma, St. Louis, MO), and List Biological Laboratories (Cat#188 & 189, List Biological Laboratories Inc. …

• Product #188L – Adenylate Cyclase Toxin, Recombinant from B. pertussis

… in the presence of both Anthrax Lethal Factor (recombinant from B. anthracis) and Anthrax Protective Antigen (recombinant from B. anthracis) of Anthrax (LIST Biological Laboratories INC, USA) in concentrations of 0.5 and 0.1 g/ml, respectively. …

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).

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

Toxin neutralization assay (TNA):

An in vitro toxin neutralizing assay (TNA) was carried out with J774A.1 mouse macrophage cell line (ECACC cat no 91051511) as done previously with modifications^24,56. The 96-well flat-bottomed tissue culture plates (Greiner bio one, Germany) seeded with 80 000 macrophages per well in Dulbeccos modified eagle media (DMEM) and 10% foetal calf serum (FCS) were incubated overnight at 37C and 5% CO₂. Test sera were diluted 2-fold (starting dilution; 1:50) in culture medium with PA and LF (List Biological Laboratories Inc., Campbell, USA) at concentrations of 500ng/mL and 400ng/mL respectively. The serum/lethal toxin mixture was incubated for 1h at 37C and 5% CO2 before addition to overnight-cultured cells and incubation for 3h. …

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).

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

Reagents:

… PA (catalog number 171E) and EF (catalog number 178a) were purchased from List Biological Laboratories (Campbell, CA).

ChIP-Seq and data processing:

PBMC were plated at 2 million cells per ml in RPMI containing 10% FBS, 100 units/ml penicillin, and 100g/ml streptomycin. These cells were then treated with 10nM ET (10nM EF and 10nM PA) or 500M 6MB-cAMP for 4.5h and then isolated by centrifugation followed by snap freezing in liquid N₂ as recommended by Active Motif. For each condition, 60 million PBMC were prepared from 3 donors (20 million PBMC per donor). ChIP was performed by Active Motif with antibodies validated for ChIP against CREB (Cell Signaling Tech; catalog number 9197) or against C/EBP (Santa Cruz Biotechnology, catalog number sc-150). After ChIP DNA libraries were constructed, 75-nt sequence reads were generated by Illumina sequencing.

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #178A – Anthrax Edema Factor (EF), Recombinant from B. anthracis

… All mice used were between 8 and 12 weeks old. Diphtheria toxin (DT) (List Biological Laboratories, Inc., Campbell, CA) was resuspended in 1X PBS and mice were injected ip with 150 ng/g DT in 100 L of PBS every 24 hours (hrs) for 3 days …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

EAE:

… Pertussis toxin (500 ng/mouse) (List Biological Laboratories) was injected intraperitoneally on days 0 and 2. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Materials and methods:

Anthrax lethal factor (Product #169L or Product #172L), MAPKKide Plus (Product #532) and the chicken IgY polyclonal anti-LF antibody (Product #769A) are products of List Biological Laboratories, Inc. …

• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #172L – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #532 – MAPKKide Plus (AMC) Specific Substrate for Anthrax Lethal Factor
• Product #769A – Anti-Lethal Factor from B. anthracis (Chicken lgY)

Sample preparation:

Fluorogenic substrates.

All stock solutions of the fluorogenic substrates were made in DMSO based on the peptide content and diluted in the appropriate optimized buffers described below. For the initial screen, all stock solutions for the substrates shown in Table 1 were 5 mM in DMSO except for LBL 10095 which was 1.6 mM in 70% DMSO. The substrates were subsequently diluted in appropriate optimized assay buffers: LBL 10079, 10081, 10097 and 10100 were diluted in 20 mM HEPES, pH 8.0 containing 0.1% Tween-20; substrate LBL 10095 was diluted in 20 mM HEPES, pH 8.0 containing 0.1% Tween-20 and 20 M ZnCl₂; substrate LBL 10108 was diluted in 20 mM HEPES, pH 8.2 containing 0.1% Tween-20.

Based on the results obtained in the initial screens, and due to its superior performance, LBL 10081, currently marketed as MAPKKide Plus, was used in all subsequent assays. Stock solutions were in 1.25 or 2.5 mM DMSO for the HPLC and the rapid microplate assays, followed by dilution in the reaction buffer 20 mM HEPES, pH 8.0 containing 0.1% Tween-20. The final concentration was 1.25 M or 2.5 M MAPKKide Plus in the reaction mixture. For the assay using the antibody-coated Nunc-Immuno Maxisorp Tubes, the MAPKKide Plus final concentration in the reaction was 10 M.

Anthrax lethal factor in plasma.

For the rapid microplate assay, given amounts of LF were spiked into neat bovine plasma and then diluted 1:10 or 1:5 in the reaction buffer. For the assays using the antibody-coated C8 Starwell plates or the immuno- tubes, the LF was added to neat bovine plasma and not diluted.

Anthrax lethal factor chicken antibodies.

For the assays using the antibody-coated C8 Starwell plates, the chicken affinity purified polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A) was diluted to 10 g/ml in 0.05M sodium carbonate-bicarbonate pH 9.6 buffer and each well was coated with 150 l of the 10 g/ml solution for a final coating of 1.5 g IgY/well. For assays using the immuno-tubes, each tube was coated with 1 mL of the same 10 g/ml solution of anti-LF antibody (List Prod # 769A). Both the plates and the immuno-tubes were incubated with the IgY overnight at 28C prior to use in the assay.

LF activity assays

Microplate assay methods.

These assays were performed using a SPECTRAmax GEMINI XS fluorescence microplate reader (Molecular Devices). The cleavage reaction was initiated by addition of the substrate.

For the initial screen of the six substrates shown in Table 1, the substrate concentration (2.5 M) was optimized to minimize background while maintaining measurable cleavage. Solutions of LF ranging from 0.5 to 2 ng/ml in 1:10 diluted bovine plasma or assay buffer were prepared. For all experiments the fluorescence was monitored at 37C hourly for 5 or 6 hours followed by an additional 18 to 18.5 hr overnight incubation at ambient temperature. For the 7-amido-4-methyl-coumarin (AMC) containing substrates, the excitation wavelength was 368 nm and emission was 452 nm with a cutoff filter at 435 nm. For the substrate containing 7-amido-4-trifluoromethyl-coumarin (LBL 10108) the excitation wavelength was 372 nm and emission was 489 nm with a cutoff filter at 420 nm. For the rhodamine-containing substrate, the excitation wavelength was 494 nm, the emission wavelength was 531 nm with a cutoff filter at 515 nm. All results are the average of duplicate wells for the samples and the average of twelve wells for the blanks.

A rapid assay method was evaluated for two ranges of LF: 10 to 1000 pg LF/ml 1:10 diluted bovine plasma and 5 to 250 ng/ml 1:5 diluted bovine plasma. Dilution of the bovine plasma was necessary in order to minimize background.

For the range 10 and 1000 pg/ml 1:10 diluted plasma, 1.25 M MAPKKide Plus was added directly to the diluted bovine plasma and the time-dependent increase in fluorescence was monitored at 37C hourly for 5 hours followed by an additional 19 hour overnight incubation at ambient temperature. The excitation wavelength was set to 368 nm and emission to 452 nm with a cutoff filter at 435 nm. The samples were run in replicates of four. For the blank samples containing no LF there were 3 sets of quadruplicates and the standard deviation was calculated from these three sets. At each time point, the plate was read 5 times to increase the precision of the fluorescence readings. The standard curve was analyzed using a linear regression fit forcing the intercept through the mean value of the blanks. The limit of detection was calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 sets of quadruplicates) and calculated as pg LF/ml plasma using the standard curve.

Subsequently, 6 data sets were evaluated, 2 data sets per day for 3 consecutive days. The data is presented as the average of these 6 data sets, each with 4 replicate samples and 12 replicate blanks. At each time point, the plate was read 3 times to increase the precision of the fluorescence readings.

For the method detecting higher levels of LF (5 to 250 ng/ml 1:5 diluted bovine plasma), 10 M MAPKKide Plus was added directly to the diluted bovine plasma, and the assays were run at 37C using the kinetic mode of the plate reader with readings at 1 or 3 minute intervals. The samples were run in triplicate with 9 replicate blanks. The limit of detection was calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 9).

A microplate assay method using antibody capture was also developed. Nunc-Immuno Maxisorp Tubes were incubated with anti-LF IgY overnight at 28C and then washed 5 times with 1.5 mL each of phosphate-buffered saline (PBS) containing 0.05% TWEEN-20 (PBST). The anti-LF coated tubes were exposed to 1 mL of a series of LF concentrations ranging from 20 to 2000 pg/ml in neat bovine plasma. The tubes were incubated at 22C for 1 hour, then washed 3 times with 1.5 ml PBST, and 1.0 ml of 10 M MAPKKide Plus was added. The reaction was allowed to proceed for 3, 5 and 22 hours at 37C. At each time point, 250 l of the reaction mixture was removed and placed in a 96-well plate. The excitation wavelength was 368 nm and emission was 452 nm with a cutoff filter at 435 nm. Any excitation wavelength between 360 to 368 nm can be used.

The experiment included triplicate tubes for each sample and nine blank tubes. The standard curve was analyzed using a linear regression fit forcing the intercept through the mean of the blanks. For the blanks there were three sets of triplicates and the standard deviation was calculated from these sets of triplicates. The limit of detection was calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 triplicates) calculated as pg LF/ml neat plasma.

HPLC assay method.

After the C8 Starwell plates were incubated with the anti-LF IgY overnight at 28C, they were washed three times with 0.1M Glycine-HCl, pH 2.5. This wash was included to remove residual LF retained with the antibody after the affinity purification and helped to minimize background fluorescence. After an additional 6 washes with PBST, the anti-LF coated wells were exposed to 300 l of a series of LF concentrations: 2.5 to 20 pg/ml neat bovine plasma, ovine plasma or milk (2% fat). The plates were incubated at 22C for 2 hours, then washed 6 times with PBST and 250 l of 1.25 M LBL 10081 (MAPKKide Plus) was added. The reaction was allowed to proceed for 2, 3.5 and 5 hours at 37C and overnight at ambient temperature (RT). At each time point 200 l of the reaction mixture was removed from replicate wells and placed in HPLC sample vials.

HPLC was performed using a Varian ProStar HPLC system (Agilent) with a Zorbax Eclipse Plus C18 reverse phase column 4.6 x 150 mm (Agilent) and a guard column containing the same resin. Solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in acetonitrile. The 16 minute HPLC method was as follows: 25% B for 0.75 minutes; 25 to 45% B in 4.75 minutes; 45 to 100% B in 0.75 minutes; 100% B for 3.75 minutes; 100 to 25% B in 0.67 minutes and 5.34 minute equilibration with 25% B. The injection volume was 20 l. The column effluent was monitored using an Hitachi fluorescence detector with excitation of 350 nm and emission of 450 nm to detect the free fluorophore, 7-amino-4-methylcoumarin, at the retention time of 4.8 minutes.

The samples were run in duplicate with 6 blank samples. The standard curve was analyzed using a linear regression fit forcing the intercept through the mean of the blanks. The limit of detection was calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 6), calculated as pg LF/ml neat plasma or milk (2% fat), from the standard curve generated at each incubation time.

… the right midcostal diaphragm muscle was exposed via a subcostal abdominal incision and injected with 5-10 µL of either 2% tetramethylrhodamine dextran (MW = 3,000 kDa; ThermoFisher, Waltham, MA) or 1% cholera toxin subunit B (CTB; List Biologicals Labs, Inc.) …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt