1. Introduction & Background
 Organic dusts represent particles accumulating from aerosols derived from animal-
vegetable- and microbial matter. Organic dusts are abundant in many workplaces
(agricultural, industrial) and in homes.
 Organic dusts can pose an environmental risk when airway/lung inflammation is
induced by inhalation of the organic dust. There are many potential pro-inflammatory
components of organic dusts. However, GNB endotoxins are thought to be particularly
important and provide a sensitive marker of microbial contamination.
 Endotoxins are unique, highly abundant, very stable, and extremely potent activators
of innate immunity in humans and other mammals, resulting in airway/lung
inflammation when inhaled.
 Endotoxins are unique glycolipids (lipopolysaccharides-LPS or lipooligosaccharides-
LOS). They are integral components of the unique outer membrane (OM) of GNB
 The bioactivity of endotoxin in mammals requires the ordered action of several host
endotoxin-recognition proteins that extract individual endotoxin molecules from the
GNB-OM and deliver endotoxin monomers to the pro-inflammatory MD-2/TLR4
receptor.
 Endotoxin is measured in clinical and environmental chemistry laboratories with the
Limulus Amoebocyte Lysate (LAL) assay: this assay is simple, sensitive (50 EU/ml-
0.005 EU/ml) and quantitative and generally distinguishes different endotoxin species
that vary in bioactivity in mammals.
 However, in contrast to the similar bioactivity in mammalian (e.g., mouse)
airways/lungs of immuno-active endotoxins added either as purified LPS/LOS, shed
OM, or intact GNB, the LAL assay underestimates bioactive endotoxin when it is
presented as part of intact GNB.
 Our preliminary data suggest that the inefficiency of the LAL assay to detect LPS of
intact bacteria can be overcome by pre-treating the bacteria with 100mM Tris-
HCl/10mM EDTA (pH 8.0), known to release abut 30%-50% of the OM LPS from intact
GNB.
Abstract
Gram-negative bacteria (GNB) found in various forms of dust contain endotoxin that can
produce several health issues (septic shock, asthma). Detection and measurement by the
kinetic chromogenic Limulus amoebocyte lysate (LAL) assay is important to properly
assess exposure in a particular environment. Due to inefficient recognition of endotoxin
intact with GNB by the LAL assay, we compared a 100mM Tris/10mM EDTA pretreatment
to pyrogen-free water (PFW), the conventional method. We used two different organic
dusts, House and Barn, and extracted them in PFW and Tris/EDTA to compare if results
from the Tris/EDTA treatment were limited to one particular dust. Tris/EDTA resulted in a
more effective treatment in improving endotoxin unit (EU/ml) detection in the LAL assay
than PFW.
Conclusions
•100mM Tris/10mM EDTA reproducibly improves detection
of bioactive endotoxin in organic dusts (Barn & House) than
the method currently used (PFW)
› BD 6-fold
› HD 3-fold
•Barn Dust has more bioactive endotoxin present than
House Dust by 8-fold
•Effect in detection of bioactive endotoxin in organic dust
was not observed with purified LPS (standard); implying the
presence of intact GNB (live or dead) in organic dusts.
Improving Detection of Bioactive Endotoxin in Organic Dusts
Dulce Chavez-Theresa L Gioannini Women in-science-Fellow
Brita Kilburg-Basnyat, Kimberly Hoppe, Peter Thorne, Jerry Weiss
Department of Sciences & Mathematics, Saint Mary-of-the-Woods College,
Saint Mary of the Woods, IN 47876
Acknowledgments
FUTURE in Biomedicine Program
Athmane Teghanemt
Environmental Health Sciences Research Center
(Grant NIH P30 ES005605)
SMWC Department of Science and Mathematics
SMWC Career Development Center
Dave Grabowski
References
1. (LONZA), P. &. (2014). Kinetic Chromogenic Limulus Amoebocyte Lysate (LAL)-
MANUAL .
2. Leive, L. (1965). A Nonspecific Increase in Permeability in Escherichia Coli Produced by
EDTA. National Institute of Arthritis and Metabolic Diseases, national Institutes of
Health, 745-750.
3. Leive, L. (n.d.). The Barrier Function of the Gram-negative Envelope. Laboratory of
Biochemical Pharmacology, 109-129.
4. Post, D. M., Zhang, D., Eastvold, S. J., Teghanemt, A., Gibson, W. B., & Weiss, J. (2005).
Membrane Transport, Structure, Function, and Biogenesis: Biochemical and
Functional Characterization of Membrane Blebs Purified from Neisseria meningitidis
Serogroup B. The Journal of Biological Chemistry, 38383-38394.
Future Work
• Are there structural differences in endotoxin present in
varying dusts?
› If yes, is 100mM Tris/10mM EDTA effective in all?
› Are contrasting endotoxin structures detected with the
same sensitivity?
• Does increase in EU in Tris/EDTA extracts reflect
increase in extraction of bioactive endotoxin or other
endotoxin-like compounds?
• Test more environmental samples in order to determine
possible change in preferred method to Tris/EDTA as the
pretreatment.
Extraction
Purification
Figure 1: Endotoxin (LPS/LOS): unique, abundant,
integral OM component of intact GNB
Figure 2: Endotoxin structure
Results
Compilation of all Data
Sample Mean n P-value
Tris/EDTA PFW Tris/EDTA PFW
Barn Dust 85,034 15,273 16 22 <0.0001
House Dust 11,378 3,639 20 20 <0.0001
Pure/LPS standard 31 28.3 3 3 0.0782
Factor C
Proenzyme
Factor C
Enzyme
Endotoxin
Factor C
Enzyme
Bound
P-nitroaniline
Substrate
Free
PNA
Factor C
Enzyme