Effect of extraction and assay media in the analysis of airborne endotoxin

Institute for Risk Assessment Sciences (IRAS), division of Environmental Epidemiology (EEPI), Utrecht University, Utrecht, the Netherlands; Business Unit Quality and Safety, division Food & Chemical Risk Analysis, TNO Quality of Life, Zeist, the Netherlands; Environmental Health Sciences Research Center, University of Iowa, Iowa City, IA, USA

^* To whom correspondence should be addressed. Email: I.M.Wouters{at}uu.nl.

	Abstract

Measurement of airborne endotoxin is thus far not standardized. Earlier studies reported higher endotoxin yields when Tween-20 was added to media used for filter extraction and in the Limulus Amebocyte Lysate (LAL) assay. This study compared four common media, and assessed the effects of Tween during extraction and analysis separately.

Parallel airborne samples from 5 work environments (n=250) were used to compare four media (pyrogen-free water (PFW), PFW-Tween-20, PFW-Tris and PFW-triethylamine-phosphate (TAP)) and extraction for 10 or 60 minutes. A subset of the extracts in PFW or PFW-Tween (n=40) were analyzed in parallel LAL assays with PFW or PFW-Tween as the assay medium.

Compared to the reference procedure (PFW, 60 min shaking), shorter extraction or presence of Tris produced similar results. Use of PFW-TAP showed overall lower yields and a deviant calibration curve. Tween in the extraction medium resulted in significantly (p<0.05) higher endotoxin yields from all dust types, independent of the effect of Tween in the assay. Tween in the LAL assay, however, also strongly inhibited the reactivity of the lipopolysaccharide (LPS) standard, thus shifting the calibration curve to higher values. Inhibition of LPS in test samples was less pronounced and varied between dust sources, resulting in enhanced calculated concentrations. This assay effect could be circumvented by diluting extracts at least 50-fold before the LAL assay.

In conclusion, of the media tested, only Tween enhances the endotoxin extraction efficiency from airborne samples in a consistent manner. We recommend extraction in PFW-Tween combined with dilution and LAL analysis in PFW.