Evaluation of the Limulus Amebocyte Lysate and Recombinant Factor C Assays for Assessment of Airborne Endotoxin

Abstract

As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory-and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) samplers, Button samplers, closed-face cassettes, and cyclone samplers. Field sampling was performed in 10 livestock production facilities, including those housing swine, chicken, turkey, dairy cows, cattle, and horses. Laboratory sampling was performed in exposure chambers using resuspended airborne dust collected in five livestock facilities. Paired samples were extracted in pyrogen-free water with 0.05% Tween 20 and analyzed using LAL and rFC assays. In 402 field sample pairs there was excellent agreement between endotoxin concentrations determined by LAL and rFC (r ‫؍‬ 0.93; P < 0.0001). In 510 laboratory sample pairs there was also excellent agreement between the two assays (r ‫؍‬ 0.86; P < 0.0001). Correlations for subgroups of facility or dust type ranged from 0.65 to 0.96. Mixed-model analysis of variance (ANOVA) for the field studies showed significant interactions of facilitysampler and facility-assay. rFC/LAL ratios of the geometric means were 0.9 to 1.14 for the samplers (not significantly different from 1.0). The data from this study demonstrate that the LAL assay and the rFC assay return similar estimates of exposure in livestock facilities. Both methods provided suitable lower limits of detection such that all but 19 of 1,824 samples were quantifiable.

Endotoxin Exposure: Predictors and Prevalence of Associated Asthma Outcomes in the United States

“…In other studies performed in large U.S. inner cities (15,16) and in a European consortium study (2), investigators reported higher endotoxin levels in bedroom floor and/or bedding (Table 5). Although endotoxin measurements in several national and regional studies were done by our laboratory, caution must be exercised when comparing endotoxin data across laboratories, owing to differences in sample collection, storage, extraction, and assay techniques (17)(18)(19)(20).…”

Optimization and Influence Mechanism of Sampling and Analysis of Airborne Endotoxin Based on Limulus Amebocyte Lysate Assay

“…PFW with Tween 20 and normal saline (NS) were frequently recommended as extraction solutions (Oppliger et al, 2005;Madsen et al, 2006;Dutil et al, 2009;Thorne et al, 2010). In this study, endotoxin concentrations were detected and evaluated under different types and different concentration gradient solution conditions.…”

The change in nasal inflammatory markers after intranasal challenges with particulate chitin and lipopolysaccharide: a randomized, double‐blind, placebo‐controlled, crossover study with a positive control

“…A 1-mL aliquot was removed and assayed for endotoxin content using the kinetic chromogenic Limulus amebocyte lysate (LAL) assay (Lonza) as described. 10 Briefly, 100 μL of the aliquot was diluted in an 8-step series of 1:10 dilutions using EF water (Lonza) in borosilicate tubes that had been depyrogenated by heating overnight at 160°C. A 12-point calibration curve was generated (r 2 = 0.999) ranging from 0.0244 to 50 EU/mL of Escherichia coli 0111:B4 control standard endotoxin calculated from the maximum slope of the absorbance vs time plot for each well.…”