A sensitive double-sandwich ELISA for neutrophil elastase. Assay results in unconcentrated bronchoalveolar lavage fluid.
We developed a sensitive double-sandwich ELISA assay for neutrophil elastase (NE) using affinity-purified NE antibody. The assay was capable of detecting NE levels of 0.2 ng/ml and was used to determine NE in bronchoalveolar lavages (BAL) of 12 healthy subjects (6 nonsmokers and 6 smokers) with a mean age of about 27 yr. NE levels in the unconcentrated cell-free supernatant of BAL, subjected to high-speed centrifugation (17,000 x g for 30 min) to sediment subcellular debris, were similar in the smokers who abstained overnight from smoking and in the nonsmokers (24.4 +/- 13.9 versus 23.7 +/- 12.3 ng/mg [SD] albumin). NE levels were significantly higher in lavage fluid not subjected to high-speed centrifugation, reflecting the presence of NE bound to subcellular debris that was sedimented by high-speed centrifugation. Concentration by ultrafiltration through a Millipore CX-10 filter was accompanied by loss of protein with a relatively greater loss of NE than albumin, resulting in lower NE/albumin ratios in concentrated than in unconcentrated lavage. It is therefore recommended that NE levels be determined on unconcentrated BAL after high-speed centrifugation to sediment subcellular debris.
Ochnio
J
J
Respiratory Division, University of British Columbia, Vancouver General Hospital, Canada.
Abboud
R T
RT
Smyrnis
E M
EM
Johal
S S
SS
D000328:Adult; D000418:Albumins / Q000032:analysis; D001992:Bronchoalveolar Lavage Fluid / Q000201:enzymology* / Q000473:pathology; D002452:Cell Count; D002498:Centrifugation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D019272:Leukocyte Elastase; D008264:Macrophages / Q000473:pathology; D009504:Neutrophils / Q000473:pathology; D010196:Pancreatic Elastase / Q000032:analysis*; D012907:Smoking / Q000378:metabolism / Q000473:pathology; D014462:Ultrafiltration
D000418:Albumins; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase