Production and Characterization of Monoclonal Antibodies against the Lethal Factor Component of Bacillus anthracis Toxin

Patterns of antibody response in humans to the anthrax vaccine adsorbed (AVA) primary (six-dose) series☆

“…Vaccination with this relatively crude preparation stimulates antibodies that bind to PA. Binding of PA impedes toxin entry into cells, and is important in neutralizing B. anthracis LT in vivo and in vitro [20]. We and others have shown that there is strong correlation between IgG to PA and toxin-neutralizing activity after human infection with virulent anthrax, and vaccination with AVA [21,22].…”

“…Samples were retrieved from −70 • C storage that met preestablished criteria: (a) Vaccinations must have occurred within defined time intervals after receipt of the initial AVA injection (day 0 [dose #1], day 14 [range [11][12][13][14][15][16][17][18][19][20][21] ). (b) Pre-injection sera were defined as those taken on the day of each vaccine dose.…”

Western blot analysis of the exotoxin components from Bacillus anthracis separated by isoelectric focusing gel electrophoresis

“…1 and 2). These mAbs were previously reported to neutralize the biological activity of the respective exotoxin [29][30][31] and were used to ensure the presence of functional epitopes on each protein.…”

“…After the transfer, nitrocellulose membranes were blocked with 5% milk and 0.1% Tween 20 in PBS before incubating with monoclonal antibodies (mAbs). To detect PA, membranes were incubated in mAb PA 14B7 [29]. To simultaneously detect LF and EF, the membranes were incubated in both mAbs LF 3F6 and EF 9F5 [30,31].…”

“…To detect PA, membranes were incubated in mAb PA 14B7 [29]. To simultaneously detect LF and EF, the membranes were incubated in both mAbs LF 3F6 and EF 9F5 [30,31]. The membranes were rinsed three times in PBST for 5 min each and then incubated with HRP-labeled goat anti-mouse IgG (Kirkegaared & Perry, Gaithersburg, MD) for 1 h. Reactive bands were detected by enhanced chemiluminescence (ECL; Amersham-Pharmacia Biotech, Piscataway, NJ) as directed by the manufacturer.…”