title: Modification of sample processing for the Limulus amebocyte lysate assay enhances detection of inflammogenic endotoxin in intact bacteria and organic dust.
abstract: The pro-inflammatory potency and causal relationship with asthma of inhaled endotoxins have underscored the importance of accurately assessing the endotoxin content of organic dusts. The Limulus amebocyte lysate (LAL) assay has emerged as the preferred assay, but its ability to measure endotoxin in intact bacteria and organic dusts with similar sensitivity as purified endotoxin is unknown. We used metabolically radiolabeled Neisseria meningitidis and both rough and smooth Escherichia coli to compare dose-dependent activation in the LAL with purified endotoxin from these bacteria and shed outer membrane (OM) blebs. Labeled [14C]-3-OH-fatty acids were used to quantify the endotoxin content of the samples. Purified meningococcal and E. coli endotoxins and OM blebs displayed similar specific activity in the LAL assay to the purified LPS standard. In contrast, intact bacteria exhibited fivefold lower specific activity in the LAL assay but showed similar MD-2-dependent potency as purified endotoxin in inducing acute airway inflammation in mice. Pre-treatment of intact bacteria and organic dusts with 0.1 M Tris-HCl/10 mM EDTA increased by fivefold the release of endotoxin. These findings demonstrate that house dust and other organic dusts should be extracted with Tris/EDTA to more accurately assess the endotoxin content and pro-inflammatory potential of these environmental samples.
authors.affiliation: 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Hoppe Parr
authors.affiliation: 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USA.
authors.forename: Suzana
authors.initials: S
authors.lastname: Hađina
authors.affiliation: 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USA.
authors.forename: Brita
authors.initials: B
authors.lastname: Kilburg-Basnyat
authors.affiliation: 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USA.
authors.forename: Yifang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USA.
authors.forename: Dulce
authors.initials: D
authors.lastname: Chavez
authors.affiliation: 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USA.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Thorne
authors.affiliation: 2 Departments of Internal Medicine and Microbiology and Inflammation Program, The University of Iowa, Iowa City, IA, USA.
authors.forename: Jerrold P
authors.initials: JP
authors.lastname: Weiss
mesh_terms: D000818:Animals; D002250:Carbon Radioisotopes; D003951:Diagnostic Errors / Q000517:prevention & control; D004391:Dust / Q000032:analysis; D004731:Endotoxins / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000276:immunology*; D006801:Humans; D008033:Limulus Test / Q000379:methods*; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009345:Neisseria meningitidis / Q000276:immunology*; D011014:Pneumonia / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D002250:Carbon Radioisotopes; D004391:Dust; D004731:Endotoxins; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96
keywords: Asthma; endotoxin; house dust; lipopolysaccharide; organic dust