Bacterial Toxin Research Citations

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

Cell culture and infection experiments:

RAW264.7 macrophage-like cells (ATCC No. TIB-71) were cultured in RPMI (10% FCS, 1% L-Glutamine) at 37C/5% CO₂. For infection experiments for analysis of LDH and Il-1 release, cells were seeded in 96 well plates the day before. 20000 RAW264.7 cells were seeded per well and were pre-stimulated with 1 g/ml E. coli LPS (List Biological Laboratories, California) overnight to induce expression of pro-IL-1. …

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

 Materials and Methods:

… The light-chain activity and cleavage pattern of these BoNT/A products were assessed using the novel LCA-HPLC method reported here with BoNT/A substrate (SNAPtide520; List Biological Laboratories, Inc.; Campbell, CA, USA). SNAPtide520 sequence: Abz Thr D-Arg Ile Asp Gln Ala Asn Gln Arg Ala Thr Lys Nle Lys Dnp; where Abz = o-Aminobenzoic acid (FRET pair fluorophore) and Dnp = 2,4-Dinitrophenyl (FRET pair quencher).

BoNT/A cleavage fragment (designated as SNAPtide529 by List Biological Laboratories, Inc.) was utilized to identify retention time of the fragment. …

Commercially available BoNT/C FRET substrate (SYNTAXtide560; List Biological Laboratories, Inc.; Campbell, CA, USA) was utilized to assess type-C cleavage. Because of the RP-HPLC reaction-product readout, no modification to the method was necessary in order to observe the reaction products generated using the SYNTAXtide560 substrate. …

• Product #520 – SNAPtide Substrate (o-Abz/Dnp) for C. botulinum Type A Neurotoxin
• Product #529 – Unquenched Calibration Peptide for SNAPtide 520 Substrate for C. botulinum Type A Neurotoxin
• Product #560 – SYNTAXtide (o-Abz/Dnp) Peptide Substrate for C. botulinum Type C Neurotoxin

Protein Purification:

Highly purified recombinant proteins were prepared in our laboratories for binding assays as well as immunizations, as described below. For ELISA and LTx neutralization assays, purified PA and LF were purchased from List Biologicals Laboratories, Inc., Campbell, CA. …

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

PTx (holotoxin), the full length PTx-S1 (1-235) protomer, and the B oligomer were purchased from List Biological Laboratories, Inc., Campbell, CA.

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)

Macrophage stimulation:

Thioglycollate-elicited peritoneal macrophages were prepared as previously described (25, 41) and stimulated with 100 ng/ml LPS (Ultra-pure; List Biological Laboratories), …

Author did not specify which List Labs LPS product was utilized in their research.  List Labs provides the following LPS products:  https://listlabs.com/product-information/lipopolysaccharides/

Materials:

SNAPtide substrate #520 contains the FRET pair o-aminobenzoic acid/2,4 dinitrophenyl (o-Abz/Dnp) and SNAPtidesubstrate Mca/Dnp contains (7methoxy-coumarin-4-yl)acetyl/2,4 dinitrophenyl as its FRET pair. Botulinum Neurotoxin Type A (Product #130A) and the SNAPtidesubstrates are products of List Biological Laboratories.

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

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

•  ELISA assay showing specific binding of BTA to SV2c
•  MagneGST-SV2c capture assay
•  MagneGST-SV2c capture assay with HPLC analysis
•  HPLC peak intensity for cleaved SNAPtideas a function of BTA

Materials:

SNAP Etide550, VAMPtide540, SNYTAXtide560, and SNAPtidesubstrates 520, 521, and 522, and the Botulinum neurotoxin types A, B, C, and E light chains, recombinant (Product #610A, 620A, 625A, and 635A) are products of List Biological Laboratories, Inc.

• Product #550 – SNAP Etide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type E Neurotoxin
• Product #540 – VAMPtide Peptide Substrate (o-Abz/Dnp) Substrate for C. botulinum Type B Neurotoxin
• Product #560 – SYNTAXtide (o-Abz/Dnp) Peptide Substrate for C. botulinum Type C Neurotoxin
• 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 #610A – Botulinum Neurotoxin Type A Light Chain, Recombinant
• Product #620A – Botulinum Neurotoxin Type B Light Chain, Recombinant
• Product #625A – Botulinum Neurotoxin Type C Light Chain, Recombinant
• Product #635A – Botulinum Neurotoxin Type E Light Chain, Recombinant

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

• Fluorimetricassays
• HPLC
• Limit of Detection

LF quantification:

… To achieve the necessary dynamic range and to account for high-sensitivity rapid-response sample analysis, two standard curves were prepared, one for 20 ng/ml, l (low volume [LV]) and the other for 200 ng/ml, l (high volume [HV]) serum volumes. In addition, three quality control samples (QCs) were prepared in human serum pools (Interstate Blood Bank, Memphis, TN) using recombinant LF (List Biological Laboratory, Campbell, CA) and validated. …

PA antigen capture immunoassay:

Microtiter plates (Thermo Labsystems, Franklin, MA) were coated with monoclonal anti-PA antibody AVR1046 obtained by the same methodologies used for anti-LF monoclonal described previously (4) at 1.28 g/ml in 0.01 M phosphate-buffered saline (PBS), pH 7.4. Coated plates were washed with a wash buffer consisting of PBS and 0.1% Tween 20, pH 7.4. Recombinant PA (rPA; List Biological Laboratories, Inc., Campbell, CA) was serially diluted twofold using a 1/25 or a 1/100 dilution of normal rhesus macaque serum (RMS). The rPA dilutions formed a seven-point standard curve from 0.6 to 37.5 ng/ml. …

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

Tracer injections

Several minutes after being given intraperitoneal injections of a cocktail, consisting of 45% ketamine (100mg/ml), 35% xylazine (20mg/ml) and 20% physiological saline at a dose of 0.16 ml/100g of body weight, rats were placed into a Kopf stereotaxic instrument. The skulls were exposed and small bore holes were created to allow selected brain structures to be targeted by filament-containing borosilicate glass pipettes (O.D. – 1.0 mm) pulled to tip diameters of 10–25 μm and containing the retrograde tracers Fluoro-Gold (FG; Fluorochrome, Inc., Englewood, CO; 1% in 0.1M cacodylate buffer, pH 7.4) or cholera toxin B subunit (CtB, List Biological Laboratories, Campbell, CA),

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