An easy microtiter assay for quantitation of cytokine induction by lipopolysaccharide (LPS) and activity of LPS-binding serum components.
Lipopolysaccharide (LPS, endotoxin) is a major inducer of cytokines, such as interleukin 1 (IL1), IL6, IL8 and tumor necrosis factor (TNF). A convenient microtiter assay was developed to measure such activity. LPS coated onto a plastic surface was used to stimulate purified human mononuclear cells (MNC) in microtiter plates. Following stimulation the supernatants were assayed for presence of TNF by ELISA. Purified rough and smooth LPS from Pseudomonas aeruginosa gave a dose-dependent TNF release over a range of 0.1-1.0 microgram LPS/well. The assay was subsequently used to investigate the biological activity of anti-LPS antibodies and other LPS-specific serum components in sera from patients with cystic fibrosis (CF). As a group, sera from 10 CF patients chronically infected with P. aeruginosa did not affect the LPS-induced TNF release, while sera from normal controls inhibited this biological activity. When individual CF patients with or without chronic lung infection are considered, the antibodies appear to either enhance or inhibit the LPS-stimulated TNF release (range: 73-120%), while all antibodies from healthy controls inhibit the activity of LPS (range: 76-97%). Only a weak correlation (rho = 0.491, p = 0.037, n = 19) was found between the antibody titer in ELISA and the biological activity of sera. This new assay is suggested for convenient measurement of interference with cytokine induction from human MNC by patient or therapeutic anti-LPS antibodies and other LPS-specific serum components.
Garde
A H
AH
Institute of Medical Microbiology and Immunology, Panum Institute, Copenhagen, Denmark.
Fomsgaard
A
A
Høiby
N
N
D000907:Antibodies, Bacterial / Q000276:immunology; D003550:Cystic Fibrosis / Q000276:immunology; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology*; D011552:Pseudomonas Infections / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D000907:Antibodies, Bacterial; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha