Citations

DC and NK Cell Depletion:

Depletion of cDCs was induced in CD11c-DTR-Gfp mice as described.35 Briefly, 100 ng (4 ng/g) of diphtheria toxin (DT) (List Biological Laboratories Inc.) was administered intraperitoneally 1 day after the last dose of CCl₄. Three days later, the mice were sacrificed and livers were harvested (Supporting Fig. 2B). NK cell depletion using anti-asialo-GM1 antibody (Wako Chemicals) and NKp46-DTR-eGFP mice were performed as described32, 36 (Supporting Fig. 2F,G).

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

Materials and Method:

Experiments using nine C57BL/6J mice were performed according to lab procedures approved by University of California at San Diego Laboratory Animal Care and Use

Animals:

Injections into the medulla used purified Cholera toxin B subunit (CTb) isolated from Vibrio cholera (Product #103b, List Biological Laboratories, Inc.). … primary antibody diluted 1:10,000 (Product #703, List Biological Laboratories, Inc.) left rotating overnight. …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)

Recording

We recorded neuronal activity with a tungsten electrode (Microprobe Inc., impedance 0.6–1.2 MOhms) attached to a glass capillary with internal tip diameters between 8 and 25 μm (Plowden and Thompson Ltd, outer diameter 0.9 mm, internal diameter 0.12 or 0.22 mm; Fig. 1A) and containing the tracer, CTb (1% low salt solution in double distilled H2O, List Biological Laboratories Inc.). …

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

Enzyme-Linked ImmunoSorbent Assay (ELISA):

96 well microtiter plates (EIR/RIA plate, Costar, Lowell, MA) were coated with 100 l rPA (List labs, Campbell, CA) at 1 g/ml in PBS overnight (o/n) at 4C. Plates were subsequently blocked with 200 l 1% BSA (in PBS containing 0.05% Tween 20, PBS-T) for two hours at 4C. …

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

TLR Stimulation and Cytokine Measurement:

For ELISA, day 5 BM-derived macrophages were plated in a 96-well tissue plate (5 104 cells per well) overnight. Titrations of TLR stimuli were added for 16 h as follows: Salmonella minnesota R595 LPS (List Biological Laboratories), …

• Product #304 – LPS from Salmonella minnesota R595 (Re)

Materials and Methods:

SNAPtide substrates (Products #521 and #523), Unquenched Calibration Peptide for SNAPtide 521 (Prod #528), BoNT/A LC (Product #610A), and BoNT/A holotoxin (Prod #130A) are products of List Biological Laboratories, Inc. …

• Product #521 – SNAPtide Peptide Substrate (FITC/DABCYL) for C. botulinum Type A Neurotoxin
• Product #523 – SNAPtide Peptide Substrate flP6(DABCYL/5-IAF) for C. botulinum Type A Neurotoxin
• Product #528 – Unquenched Calibration Peptide for SNAPtide 521 Substrate for C. botulinum Type A Neurotoxin
• Product #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant
• Product #130A – Botulinum Neurotoxin Type A from Clostridium botulinum

Methods – The above materials were used in the following (refer to poster for details):

• Fluorimetric Assay
• Buffer Optimization
• Linearity
• Sensitivity and LOD
• Kinetic parameter evaluations
• Inner Filter Effect Correction

Reagents:

SNAPtidesubstrate (Product # 520) and Botulinum Neurotoxin Type A (Product #130A) are products of List Biological Laboratories.

Summary of Results: The His6-SV2c-His6 was purified in a soluble form and linked to magnetic nickel coated beads.  Various buffer conditions and binding conditions were tested to optimize the binding and limit the non specific binding of toxin to the beads.  We are able to detect cleavage of SNAPtideafter exposure to as little as 5pg of BoNT/A.

Conclusions:  Our study shows the ability to purify a soluble form of the SV2c BoNT/A binding domain and its utility in the capture and detection of BoNT/A.  This is a key step in establishing an in vitropotency assay.  This assay can also be used to screen for inhibitors that interfere in the binding of BoNT/A to the SV2c protein receptor.

• Product #520 – SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin
• Product #130 – Botulinum Neurotoxin Type A from Clostridium botulinum

Materials:

SNAPtide substrate (Product #520), Botulinum neurotoxin type A (Product #130A), and the chicken IgY polyclonal anti-BoNT/A antibody (Product #730A) are products of List Biological Laboratories, Inc.

• Product #520 – SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin
• Product #130A – Botulinum Neurotoxin Type A from Clostridium botulinum
• Product #730A – Anti-Botulinum Neurotoxin, Type A (Chicken lgY)

Methods – The above materials were used in the following (refer to poster for details):

• HPLC
• BoNT/A capture by the polyclonal antibody

… Commercially available protein toxin samples were used. … while shiga toxin 1 (recombinant product) was obtained from List Biological Laboratories, Inc. (CA). …

• Product #161 – Shiga Toxin 1 from Escherichia coli

Protein expression and purification:

PTx (holotoxin) in glycerol was purchased from List Biological Laboratories, Inc. (Campbell, CA). …

PTx toxoiding and characterization:

To detoxify PTx, the protocol developed by Fowler et al., similar to that used in current acellular vaccine formulations, was employed (11). Lyophilized, salt-free PTx (List Labs) was dissolved in phosphate-buffered saline (PBS) with 0.5% (wt/vol) formaldehyde and 0.05 M lysine for a final PTx concentration of 0.5 M. …

• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)