Tetramethylbenzidine substrate

ELISA was performed using standard protocols. Briefly, maxisorp surface ELISA plates (Nalge Nunc International) were coated with 50 μl of 200-ng antigen in carbonate-bicarbonate buffer/well and incubated at 4 °C overnight. Wells were aspirated and then blocked with PBS–1% bovine serum albumin at 250 μl/well at room temperature for 2 h. The blocking reagent was aspirated, and the plates were washed 5 times with PBS 0.1% Tween 20 plus 0.01% benzalkonium chloride. One hundred μl of human serum diluted in PBS at 1/100 was added per well. Samples were incubated at room temperature for 60 min. The plates were washed and 50 μl of protein A-horseradish peroxidase conjugate at a 1:20,000 dilution in PBS was added per well and incubated for 30 min at room temperature. The conjugate was aspirated, and the plates were washed. One hundred microliters of tetramethyl benzidine substrate (Kirkegaard & Perry Laboratories)/well was added and incubated for 15 min at room temperature, and the reaction was stopped with 100 μl of 1 N H2SO4/well. The plates were then read at 450 nm using an ELISA reader (ELX 808; Bio-TEK Instruments, Inc.). The cutoff for the assays was the mean of the 6 normal human serum samples plus three standard deviations of the mean.

Maxisorp immunoplates (Thermo Fisher Scientific) were coated with NP₅₀-BSA (Biosearch Technologies) or antibodies against mouse Ig isotypes, followed by blocking with 0.5% BSA in PBS containing 0.05% Tween-20. Serially diluted sera were added to the wells, followed by incubation for 5–6 h at room temperature. After washing with PBS containing 0.05% Tween-20, HRP–conjugated antibodies against mouse Ig isotypes (SouthernBiotech) were added to the wells, followed by overnight incubation at 4°C. The HRP activity was detected with tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories), and absorbance at 450 nm was measured using an iMark microplate reader (Bio-Rad) after quenching the reaction with 1 N HCl.

Microtiter plates were coated overnight with mAb F26G3 (1 μg/ml) in PBS, washed with PBS-Tween (PBS containing 0.05% Tween 20), and blocked for 90 min with PBS-Tween. Samples of plasma or urine were serially diluted in PBS-Tween and incubated for 90 min with the mAb-coated wells. Plates were washed with PBS-Tween, incubated for 90 min with horseradish peroxidase-labeled mAb F26G3 (1 μg/ml) diluted in PBS-Tween with 0.05% milk, washed and then incubated with tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The reaction was stopped after 30 min with a solution of 1 M H₃PO₄ and plates were read at an optical density of 450 nm (OD₄₅₀). Purified B. anthracis Pasteur PGA diluted in PBS-Tween was used as a standard for calculating PGA levels in rabbit plasma or urine. The concentration of purified PGA standard that produced an OD of 0.5 in a log-log plot of OD₄₅₀ vs. ng PGA/ml was calculated as the endpoint and used to determine PGA levels in the plasma and urine. The lower limit of detection of the PGA ELISA, calculated as a 3-fold increase over background, was 40 pg/ml.