Comparison of four procedures for measuring elastase production by Pseudomonas aeruginosa strains from cystic fibrosis patients.
Forty-five Pseudomonas aeruginosa strains were isolated from the sputa of cystic fibrosis patients. The elastase production of each strain was assayed in the culture supernatant using four different procedures, i.e. two immunological assays (RIA and ELISA), and two enzymatic assays, the latter employing either elastin or tetraalanine as substrate, with conductometric measurement of substrate hydrolysis. Elastase concentrations were determined from standard curves prepared with the same purified elastase, and expressed in mg of elastase per litre of supernatant. The resulting values were in the range reported in the literature, and differed greatly from one strain to another (0-230 mg/l). Linear relationships were found when assays were compared in pairs. Significant correlation coefficients were obtained (r greater than 0.76, p less than 0.001) but the values were quite different for different assays. Thus, ELISA measurements were always from three to five times higher, and RIA results were from two to five times lower, than those from the other assays. Enzymatic assays with elastin gave higher values than those using tetraalanine. Most P. aeruginosa strains produce two other proteinases, alkaline proteinase and Las A protein. Both enzymes have limited elastolytic and peptidasic activities. The presence of alkaline proteinase does not result in falsely elevated elastase values, but an increase of elastase activity was observed when Las A was preincubated with elastin. Since this increase was not observed when tetraalanine was used as the substrate, the presence of Las A in the supernatants could explain the differences observed between the enzymatic assays. The assay with the synthetic substrate is therefore preferred.
Saulnier
J
J
Laboratoire de Biochimie Analytique, Université Claude Bernard-Lyon I, Villeurbanne, France.
Wallach
J M
JM
Döring
G
G
Malissard
M
M
Vacheron
M J
MJ
Guinand
M
M
D003550:Cystic Fibrosis / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006868:Hydrolysis; D008722:Methods; D010196:Pancreatic Elastase / Q000032:analysis / Q000096:biosynthesis*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000302:isolation & purification; D011863:Radioimmunoassay; D013183:Sputum / Q000382:microbiology
D010196:Pancreatic Elastase