Pseudomonas aeruginosa Compared with Escherichia coli Produces Less Endotoxemia: Methods (4)
The concentrates and a purified recombinant exotoxin A standard (gift of Dr. David J. FitzGerald, Laboratory of Molecular Biology, National Cancer Institute) were electrophoresed on a 10 percent polyacrylamide gel followed by overnight electrophoretic blotting (Bio-Rad, Rockville Centre, NY) onto nitrocellulose membranes (Schleicher and Schuell, Inc, Keene, NH) using a pH of 8.3, 250 mmol/L of Tris, 192 mmol/L of glycine, and 20 percent methanol transfer buffer. Blots were incubated with rabbit anti-exotoxin A hyperimmune serum (gift of Dr. David J. FitzGerald) at a 1:250 dilution, followed by goat anti-rabbit antibody conjugated to horseradish peroxidase (1:1,000 dilution), and finally developing solution (Bio-Rad, Rockville Centre, NY). This method is able to detect 10 ng/ml of exotoxin A in broth supernatant. ventolin inhalers
A quantitative chromogenic LAL assay (Whittaker M.A. Bioproducts, Walkersville, MD), sensitive to 10 pg/ml of US standard endotoxin, was used to determine endotoxin concentrations as previously described. Serial blood samples (3 ml each) were collected in sterile, pyrogen-free glass tubes containing heparin (2 USP units/ml of blood) at baseline and days 1, 2, and 10 after surgery. These time points were chosen to coincide with maximal depression and full recovery of myocardial dysfunction as determined from previous studies. The P aeruginosa-infected dogs and four of the E co/i-infected dogs (14 X 10s CFU/kg) also had endotoxin determinations at 6 and 12 hours after clot implantation.