Immunoenzymometric Assays for Alkaline Protease and Exotoxin A from Pseudomonas aeruginosa: Development and Use in Detecting Exoproteins in Clinical Isolates from Patients with Cystic Fibrosis

Abstract

JafFar-Bandjee et al.: Exoproteins of Pseudomonas aeruginosa: Immunoassays in cultures from cystic fibrosis sputa 893Eur. J. Clin. Chem. Clin. Biochcm. Vol. 32, 1994, pp. 893-899 © 1994 Summary: Using immunoenzymometric assays, the production of elastase, alkaline protease and exotoxin A was determined in culture supernatants of 35 strains of Pseudomonas aeruginosa isolated frorn patients suffering from cystic fibrosis. The assays were simple, specific, sensitive and reproducible, and permitted the determination of low levels of exoproteins. A large strain variability of exoprotein production was found. Most of the strains secreted all three exoproteins, but six out of the 35 strains (17%) did not secrete at least one of the three (< 0.3 §/1). A significant correlation was observed between elastase and exotoxin A productions (r = 0.697, p < 0.001). Introductioii

Reliable Enzyme‐Linked Immunosorbent Assay Systems for Pathogenic Factors of Pseudomonas aeruginosa Alkaline Proteinase, Elastase, and Exotoxin A: A Comparison of Methods for Labeling Detection Antibodies with Horseradish Peroxidase

“…The analytical detection limits for our alkaline proteinase, elastase, and exotoxin A ELISA systems were 18 pg/ml, 34 pg/ml, and 22 pg/ml, respectively; these values are lower than those for the ELISAs described by Jaffar-Bandjee et al (16,18) (0.3 ng/ml for alkaline proteinase; 0.26 ng/ml for elastase; and 0.25 ng/ml for exotoxin A) and the ELISA described by Elsheikh et al systems is only one-tenth of that used in the previously described systems. The working range of each of our ELISA systems is 0-100 ng/ml, which is 5 times wider than that of the ELISA systems described by JaffarBandjee et al (16,18) (0-20 ng/ml) and 25 times wider than that of the exotoxin A ELISA system described by Ogaard et al (27) (0-4 ng/ml). The single most important advantage of our ELISA systems is the linearity of the standard curves within the range of 0-100 ng/ml; this facilitates easier analysis in every respect.…”

“…The single most important advantage of our ELISA systems is the linearity of the standard curves within the range of 0-100 ng/ml; this facilitates easier analysis in every respect. The standard curves of the ELISA systems described by Jin et al (20) and Jaffar-Bandjee et al (18) were nonlinear even on a logarithmic scale. The production of alkaline proteinase and exotoxin A was observed in almost all the tested strains.…”

“…Of the 2 labeling methods compared, the maleimide-pyridyl disulfide method exhibited higher sensitivity. For the preparation of IgG-HRP conjugates, Jin et al (20), Jaffar-Bandjee et al (16)(17)(18), and Schultz et al (32) used the periodate method (34). In the periodate method, the carbohydrate shell of HRP is oxidized at pH 4-5.…”

“…The analytical detection limit was defined as the concentration of each antigen calculated from the standard curve at the absorbance corresponding to the mean plus 2 SD absorbance found for the 0 calibrator (n ϭ 8), described by Jaffar-Bandjee et al (18). The analytical detection limits of each ELISA system for alkaline proteinase, elastase, and exotoxin A were 18 pg/ml, 34 pg/ml, and 22 pg/ml, respectively.…”

Mucosal and systemic antibody responses to potentialPseudomonas aeruginosavaccine protein antigens in young children with cystic fibrosis following colonization and infection

“…[18][19][20] Several P. aeruginosa antigens invoke the characteristic rise in antibody titers as the disease state progresses and can be detected in the sera, sputa, saliva, tears and bronchoalveolar lavage (BAL) fluid from CF patients. [21][22][23][24][25][26][27] Specific antibody responses to various P. aeruginosa antigens have been studied in the sera of adult patients, however, the characterization of antibody responses in children who differ in their pulmonary clinical status during the early years of life and initial stages of infection has not been conducted. [28][29][30] A study investigating serum antibodies against alkaline phosphatase, elastase and exotoxin A in 183 CF patients (mean age 16.7 y) indicated that regular determination of serum antibody may be a useful indicative measure of probable infection for CF patients with negative or intermittent P. aeruginosa cultures.…”