Radioimmunoassay for Leukotriene B4
Leukotriene B4 was measured in HPLC fractions and extracts of BAL supernatants by radioimmunoassay (LTB4 antiserum, Advanced Magnetics, Cambridge, Mass). The detection limit of this assay is 8.2 picogram (pg)/sample.
Phospholipase A, Assay
Phospholipase A activity was assayed by the method ofPatriciarca et al, as modified by Vadas. Escherichia coli strain 011 was grown in brain-heart infusion media containing l-[C]-oleic acid (New England Nuclear). Reaction mixtures included 250 p.1 E coli suspension, 250 |il 20 percent bovine serum albumin, 500 p.10.1 M tris-hydrochloride, pH 8.5, containing 10 mM CaCl and 250 |il BAL supernatant. The reaction mixture was incubated for 15 min at 37°C in an HBI vortex evaporator (Buchler Instruments, Fairfield, NJ). The reaction was stopped by cooling samples to 4°C. The cooled reaction mixture was then filtered through a 0.22 |im filter membrane to separate albumin-bound released free fatty acids from undigested l-[l4C]-oleic acid-labeled membrane-bound phospholipids. Radioactivity in the filtrates was quantitated by liquid scintillation counting. Counts were adjusted by subtraction of appropriate background and blank controls. Activity is reported as disintegrations per minute liberated from l-[l4C]-oleic acid per 250 |il BAL per 15 min. Phospholipase A activity in five of the BAL specimens used here (No. 26,29,30,32, and 33) previously has been reported.