Bacterial Toxin Research Citations

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)

HPLC Method to Assay the ADP – Ribosyltransferase Activity of Cholera Toxin

Materials: Cholera Toxin (Prod # 100B) and CTB (Prod # 104) are products of List Biological Laboratories, Inc.

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae
• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt

Materials:

The endotoxin (LPS) from E. coli K12 LCD25 was purchased from List Biological Laboratories. All LPS preparations from List Biological Laboratories, Inc., are essentially free of nucleic acid and protein and are chemically characterized with respect to their phosphate and 2-keto-3-deoxyoctonate (KDO) contents. For these studies, the purchased LPS preparation was further purified by a phenol re-extraction method to eliminate residual protein contamination prior to use in our experimental models. …

• Product #314 – LPS from Escherichia coli K12 strain LCD25

In vitro characterization of plant-produced mAbs:

ELISA was performed as previously described in reference 36, with slight modifications. Plates were coated with 1 g/mL of recombinant B. anthracis PA (List Biological Laboratories Inc., Campbell, CA). …

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

Materials:

SNAPtide substrates (Products #520 and #521), the Control Peptide for SNAPtide #520 (Product #525) and Botulinum neurotoxin type A light chain (Product #610A) are products of List Biological Laboratories, Inc.

• Product #520 – SNAPtide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin
• Product #521 – SNAPtide Peptide Substrate (FITC/DABCYL) for C. botulinum Type A Neurotoxin
• Product #525 – Control Peptide for SNAPtide 520 Substrate for C. botulinum Type A Neurotoxin
• Product #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant

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

• Fluorimetric assays
• Determination of IC₅₀ and mode of inhibition

Materials:

SNAPtide  substrate (Product #520) and Botulinum neurotoxin type A (Product #130A) 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

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

• BoNT/A capture using the F1-2 monoclonal antibody
• BoNT/A capture using the F1-40 monoclonal antibody

CYTOKINE PRODUCTION ASSAY AND MEASUREMENT OF INTERLEUKIN-1 AND TNF-:

… LPS (List Biological Laboratories, cat #201) was diluted in assay medium. Assay medium consisted of RPMI 1640 (Invitrogen Life Technologies Corporation, cat #11875) containing 1 penicillin/streptomycin/glutamine (Invitrogen, cat #10378) and 0.1% human serum albumin (Bayer AG, cat #69220). Cultures of 50% whole blood/50% assay medium in a final volume of 200 L were incubated for 18 h at 37 C and 5% CO₂ in the presence of 0 or 100 g/L LPS in a 96-well tissue-culture plate (BD Biosciences, cat #353072). LPS stimulation was initiated <1 h after blood draw and samples were run as triplicates. …

• Product #201 – LPS from Escherichia coli O111:B4