Materials and Methods
Anthrax lethal factor (Product #169), and the chicken IgY polyclonal anti-LF antibody (Product # 769A) are products
of List Biological Laboratories, Inc. The C8 Starwell Maxi Nunc-Immuno Module Plates (cat# 441653) used for LF
antibody coating and the dimethyl sulfoxide (DMSO) (cat # TS-20684) were purchased from ThermoScientific. The
96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning (cat # 3991).
Bovine plasma (cat # 7310806) and sheep plasma (cat# 7319006) were purchased from Lampire Biological
Laboratories. The milk (2% Lucerne) came from the local market.
Sample Preparation: Stock solutions of the fluorogenic substrate, MAPKKide® Plus, were 1.25 mM in DMSO
based on the peptide content determined by elemental analysis. The substrate was diluted in assay buffer:
20 mM HEPES, pH 8.0 containing 0.1% Tween-20. For the microplate assays, the LF was dissolved in neat bovine
plasma and diluted 1:10 in assay buffer. For the HPLC assays, the LF was added to neat bovine, sheep plasma or
2% milk without dilution.
LF Activity Assays:
Microplate reader: Assays were performed on a SPECTRAmax GEMINI XS fluorescence microplate reader
(Molecular Devices). The cleavage reaction was initiated by addition of the substrate, MAPKKide® Plus. The
concentration was optimized to minimize background fluorescence while maintaining measureable cleavage. For
all experiments the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 or 6 hours followed
by an additional 18 to 18.5 hr 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.
HPLC: The C8 Starwell Maxi Nunc-Immuno Module Plates were coated with 150 μl of a 10 μg/ml solution of
a chicken affinity purified polyclonal IgY antibody to anthrax lethal factor (List Prod # 769A). Plates were incubated
with the IgY overnight at 2-8°C and washed three times with 0.1M Glycine-HCl, pH 2.5. This wash was included to
liberate residual LF retained after the affinity purification of the antibody and was necessary to minimize the background. After 6 washes with PBS containing 0.05% TWEEN-20 (PBST), the anti-LF coated wells were exposed to
300 μl of a series of LF concentrations in neat plasma. The plates were incubated at 22°C for 2 hours. Plates were
then washed 6 times with PBST and 250 μl of 1.25 μM MAPKKide® Plus was added. The reaction was allowed to
proceed for 2, 3.5, and 5 hours at 37°C and overnight at ambient temperature. At each time point 200 μl of the
reaction mixture was removed from replicate wells and placed in HPLC sample vials.
HPLC was performed using a Zorbax Eclipse Plus C18 reverse phase column, 4.6 x 150 mm (Agilent) and a guard
column containing the same resin in a Varian ProStar HPLC system (Agilent). 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 column effluent was monitored using an Hitachi fluorescence detector
with excitation set to 350 nm and emission at 450 nm to detect the free fluorophore cleaved from MAPKKide® Plus.
The injection volume was 20 µl. The 7-amido-4-methylcoumarin (AMC) peak retention time was 4.8 minutes.
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Product #769B – Anti-Lethal Factor from B. anthracis (Chicken lgY)
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor