Citations

Cholera toxin (CT) was purchased from List Biological Laboratories Inc. (Campbell, CA, USA).

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

The following FRET peptides have been designed at List Biological Laboratories as substrates for the botulinum toxin enzymes. SNAPtide, Product #520 and #521 (U.S. Patent, No. 6,504,006 ) is readily recognized and cleaved by the Botulinum toxin type A (BTA), Product #130, and the BTA light chain (LcA), Product #610A. One of the substrates, Product #520, contains an oAbz/DNP FRET pair and the other, Product #521, a FITC/DABCYL FRET pair. Representative data are given below. VAMPtide, Product #540 is readily recognized and cleaved by the Botulinum toxin type B light chain (LcB), Product #620A. This FRET substrate contains an oAbz/DNP FRET pair. SNAP Etide, Product # 550 is readily recognized and cleaved by the Botulinum toxin type E light chain (LcE), Product #635A. This FRET substrate contains an oAbz/DNP FRET pair.

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

(LF) is the enzymatic component of anthrax lethal toxin which specifically cleaves the MAPK-kinase proteins. LF is also an ideal target for therapeutic inhibitors. FRET substrates for LF, MAPKKide, Product #530 and 531, have been designed at List Biological Laboratories. One of the substrates, Product #530, contains an oAbz/DNP FRET pair and the other, Product #531, a FITC/DABCYL FRET pair. Product #531 is especially well suited for high throughput screening for the IC50 and inhibitor modality of potential inhibitors.  …

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

Materials – 

SNAP-Etide^TMsubstrate (Product #550) and botulinum neurotoxin type E light chain, recombinant (Product #635A), are both products of List Biological Laboratories, Inc.

Methods – 

Fluorimentric Assay:

Continuous assays were performed on a SPECTRA max GEMINI XS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) using Greiner FLUO-TRAC black flat-bottomed plates (E&K Scientific, Campbell, CA).  Stock solutions of the FRET substrate was made in dimethylsulfoxide (DMSO).  Final dilutions were made in the appropriate buffer.  Plates were equilibrated at 37C for 15 min prior to initiation of the reaction.  For all experiments the time-dependent increase in fluorescence intensity was monitored at 37C. The excitation wavelength was set to 321 nm and emission to 418 nm. 

Buffer Optimization:

FRET assays were performed to test the activity of LcE with SNAP-Etide^TMas a function of pH, Tween-20 and ZnCl₂.  Three separate experiments were performed (Figure 1).  The cleavage reaction was initiated with addition of 5 nM LcE to the wells containing 10 M SNAP-Etide^TM in the appropriate buffer.  Initial velocities of cleavage in RFU/sec were evaluated and compared for each assay in order to determine the optimum buffer conditions for the reaction.

LcE Titration:

LcE titration experiment was performed in 50 mMHEPES, pH 7.8, 0.1% Tween-20, using 10 M SNAP-Etide^TM.  LcE was prepared at 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 M concentrations.  Following equilibration, the cleavage reaction was initiated with addition of 10 M SNAP-Etide^TM.  Initial velocities of cleavage were plotted against LcE concentration (Figure 2).

Trypsin Digest: 

Dilutions of SNAP-Etide^TMwere prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve 70, 60, 50, 40, 30, 25, 15, 7.5, 3.75, 1.88, and 0.94 M concentrations.  The reaction was initiated with addition of 10 nM trypsin into each well.  End point readings were taken after 50 min.  A second round of 10 nM trypsin was added to each well in order to achieve total enzyme digestion.  The maximum fluorescence reached was graphed as RFU/5000 against SNAP-Etide^TM concentration (Figure 3A).  An identical experiment was run using 2.5 nM LcE for digestion of SNAP-Etide^TM.  Initial velocities of cleavage were graphed in RFU/sec against substrate concentration (Figure 3B).

Inner Filter Effect Correction: 

Dilutions of SNAP-Etide^TM were prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve concentrations ranging from 250 M to 2 M.  Fluorescence end point readings of SNAP-Etide^TM at each concentration were recorded.  In order to determine theinner filter effect at each substrate concentration another set of end point fluorescence (RFU) readings were recorded after addition of 5.0 M free o-Abz-Lys.  Fluorescence intensity obtained for SNAP-Etide^TM was then subtracted from the fluorescence intensity obtained for SNAP-Etide^TMand o-Abz-Lysin order to obtain fluorescence for the free o-Abz-Lyspeptide.  The decrease in fluorescence of the o-AbzLysin the presence of SNAP-Etide^TM reflects the inner filter effect (Table 1).  A correction factor is obtained for each SNAP-Etide^TM concentration: 

correction factor = RFU (o-Abz-Lys) at each [SNAP-Etide^TM] RFU (o-Abz-Lys)

Initial reaction rates were obtained for each substrate concentration after addition of 2.5 nM LcE.  The rates were corrected as given in Table 1.  The plots of initial velocity versus SNAP-Etide^TM concentration (Figure 4)indicates a decreasing rate of cleavage at concentrations of substrate greater then 100 M.  This is consistent with substrate inhibition.  The kinetic data was analyzed using the substrate inhibition equation from Kaleida Graph software: 

ax b+(x(1+x/c)) , where a = Vmax, b = Km, and c = Ki, competitive inhibition constant

• Product #550 – SNAP-Etide Peptide Substrate (o-Abz/Dnp) for C. botulinum Type E Neurotoxin
• Product #635A – Botulinum Neurotoxin Type E Light Chain, Recombinant

Lethal Factor protease assay:

Lethal Factor (20 nM final concentration) and MAPKK substrate (MAPKKide 12.5 M, final concentration) were purchased from List Biological Laboratories, Campbell, CA and used according to the fluorescence resonance energy transfer (FRET) method. …

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 #530 – MAPKKide Peptide Substrate (o-Abz/Dnp) for Anthrax Lethal Factor

• Product #531 – MAPKKide Peptide Substrate (DABCYL/FITC) for Anthrax Lethal Factor

Sandwich ELISAs for LF detection:

Recombinant PA₈₃ and PA₆₃ were purchased from List Laboratories (New Jersey). PA₆₃ is a cleavage product that is capable of binding LF (45). For the sandwich ELISA, 50 l of PA at 63 kDa and 83 kDa (6 g/ml) was applied to a 96-well plate and blocked with 2% milk-PBS as described above. For initial assays, LF was diluted in PBS or human serum at 5 g/ml. For assays detecting LF in infected animals, serum was added to the plate initially diluted 1:1 in 2% milk-PBS and then serially diluted across the plate in duplicate. After a 1-h incubation, the plate was washed as described above. Goat anti-LF polyclonal serum (List Labs) was diluted 1:1,000 in 2% milk-PBS and added to the plate for a 1-h incubation in duplicate. The plate was then washed, followed by the addition of goat anti-rabbit IgG-HRP conjugate (Bio-Rad) diluted in 2% milk-PBS for 1 h. ELISA reactions were developed with OPD tablets (Sigma) and quenched by the addition of 50 l of 4.5 M H₂SO₄. …

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis
• Product #771B – Anti-Protective Antigen from B. anthracis (Goat)

Reagents and antibodies:

Recombinant anthrax PA and LF were purchased commercially and were stored in 1:1 glycerol-water at 20C (List Biological Laboratories) for in vitro studies. Unless otherwise indicated, anthrax LT was administered in excess at concentrations of 2.5 g/ml PA and 1 g/ml LF. In selected experiments a proteolytically inactive mutant of LF was used as a negative control (E687C substitution in zinc binding site that eliminates enzymatic activity; List Biological Laboratories). …

Anthrax PA binding assays:

Purified murine or human B cells were cultured at 4C for 30 min with FITC-labeled anthrax PA (50 g/ml; List Biological Laboratories) in the presence or absence of unlabeled anthrax PA (150 g/ml) to confirm specific binding. Stained cells were then washed with PBS and analyzed by flow cytometry (see below). Unstained cells were analyzed in parallel to establish background levels of autofluorescence.

ELISA:

Primary B cells were cultured in complete RPMI for 4 to 5 h, washed with RPMI 1640, and then stimulated as indicated in the presence or the absence of anthrax LT for 7 days.  …

Murine in vivo studies:

Mice were treated with varying doses of anthrax LT as indicated, using a fixed ratio of LF/PA of 1:2.5. LF and PA were resuspended in PBS and injected i.p. into mice in a total volume of 1.0 ml of PBS. As a negative control, selected mice were treated with PBS alone. Mice were sacrificed 3 h after treatment, and spleens were harvested for primary B cell isolation as previously described. Primary B cells were then evaluated for proliferation and IgM production.

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
• Product #176 – Anthrax Lethal Factor (LF), Recombinant, Mutant E687C from B. anthracis
• Product #175 – Anthrax PA 63 – FITC Conjugate

Intranasal immunization with killed S. aureus.

Mice were immunized with an acapsular mutant of S. aureus Reynolds designated JLO22 (7) that was cultivated in TSB to the logarithmic phase of growth (A650, 0.34). The bacteria were pelleted, suspended in phosphate-buffered saline at a concentration of 108 CFU/ml in an open petri dish, and exposed to a UV light source that was 8 cm away for 10 min on a rotator in the dark. The bacteria were concentrated by centrifugation, and 10 μl of the UV-killed bacterial suspension containing 108 CFU S. aureus with or without 5 μg of cholera toxin B (CTB) (List Biological Laboratories, Inc., Campbell, Calif.) was applied to each mouse nose on days 0, 5, and 10. CTB was omitted from the third immunization to reduce nonspecific protection. Two weeks after the third immunization, the mice were inoculated with 108 CFU S. aureus strain Newman cultivated in TSB to the logarithmic phase (A650, 0.34). Colonization was evaluated after 14 days.

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

PGRP-S secretion:

Granulocyte-enriched populations of bovine peripheral leukocytes were purified as described above and suspended to 1.2 10⁷ cells/ml in HBSS (137 mM NaCl, 5.6 mM glucose, 5 mM KCl, 4 mM NaHCO₃ 1 mM CaCl₂, 0.5 mM MgCl₂, 0.4 mM KH₂PO₄, 0.4 mM Na₂HPO₄, and 0.4 mM MgSO₄ (pH 7.4)). Aliquots of the cell suspension were incubated for 60 min at 37C in a final volume of 500 l containing one of the following stimulants: 100 nM PMA, 20 g/ml lipoteichoic acid (LTA) from Bacillus subtilis, 160 g/ml muramyl dipeptide (MDP) (Sigma-Aldrich), 100 g/ml LPS from Staphylococcus typhimurium (List Biological Laboratories), …

• Product #225 – LPS from Salmonella typhimurium

… Flat bottom microtiter plates (Nunc F96 Maxisorp) were coated with 50 l of Bacillus anthracis Protective Antigen (PA) or Lethal Factor (LF)(List Biological Laboratories (Campbell, Calif.) at a concentration of 1 g/mL in PBS overnight at 4 C. …

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

Materials:

Botulinum Neurotoxin Type A (Prod #130A) and the recombinant light chain of the Type A neurotoxin (Prod #610A) are products of List Biological Laboratories.