In brief, a 96-well microtiter plate was coated with 60 |j,L of DBA (6 |j,g/mL in phosphate-buffered saline solution [PBS]) and incubated at room temperature for 2 h. After rinsing with high-salt PBS (PBS containing 0.5 mol/L NaCl and 0.1% Tween 20), the plate was exposed to sample buffer and incubated at room temperature for 1 h. This was then treated with 50 |j,L of DBA conjugated with horseradish peroxidase (0.25 |j,g/mL) in PBS containing 1% bovine serum albumin. Before and after this setup, this plate was washed with high-salt PBS. Tetramethylbenzidine, 150 |j,L (0.42 mmol/L), in citrate-acetate buffer (pH 6.0) was added to each well and incubated for 10 min. The reaction was stopped by adding 50 |j,L of H2SO4 (normal, 4.7), Color development was read as the difference in adsorbance at 450 nm and 650 nm. The concentration of mucin was calculated by comparison with standard mucin (asialo ovine submaxillary mucin: 20 to 200 ng/mL). The amount of mucin secreted was expressed mucin weight per trachea tissue weight (nanograms per gram) Tissue weight was used as a more accurately measured surrogate for secretory surface area. comments
Lysozyme in a bacteriolytic enzyme. It has been reported that its only source in the ferret airway is the serous cells of submucosal glands. Lysozyme activity was determined spec-trophotometrically by measuring the initial rate of lysis of a 1.38 mg/mL Micrococcus lysodeikticus suspension. A 0.1-mL volume of sample buffer was added to 1.4 mL of substrate in PBS buffer at pH 6.0. The change in turbidity was measured at 540 nm. One unit of enzyme activity was defined as the change in adsorbance per minute equivalent to that produced by 1.0 mg of egg white lysozyme under identical assay conditions. The enzyme activity of the sample was calculated by comparison with standard (egg white lysozyme, 0.5 to 6 |j,g/mL).