Journal article
Modification of sample processing for the Limulus amebocyte lysate assay enhances detection of inflammogenic endotoxin in intact bacteria and organic dust
Innate immunity (London, England), Vol.23(3), pp.307-318
04/2017
DOI: 10.1177/1753425917694084
PMCID: PMC5814115
PMID: 28359219
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 [
C]-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.
Details
- Title: Subtitle
- Modification of sample processing for the Limulus amebocyte lysate assay enhances detection of inflammogenic endotoxin in intact bacteria and organic dust
- Creators
- Kimberly A Hoppe Parr - 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USASuzana Hađina - 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USABrita Kilburg-Basnyat - 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USAYifang Wang - 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USADulce Chavez - 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USAPeter S Thorne - 1 Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA, USAJerrold P Weiss - 2 Departments of Internal Medicine and Microbiology and Inflammation Program, The University of Iowa, Iowa City, IA, USA
- Resource Type
- Journal article
- Publication Details
- Innate immunity (London, England), Vol.23(3), pp.307-318
- Publisher
- United States
- DOI
- 10.1177/1753425917694084
- PMID
- 28359219
- PMCID
- PMC5814115
- ISSN
- 1753-4259
- eISSN
- 1753-4267
- Grant note
- P30 ES005605 / NIEHS NIH HHS R01 AI059372 / NIAID NIH HHS
- Language
- English
- Date published
- 04/2017
- Academic Unit
- Civil and Environmental Engineering; Occupational and Environmental Health; Pathology; Internal Medicine
- Record Identifier
- 9983997365002771
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