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. 2011 Jun;13(6):1739-45.
doi: 10.1039/c1em10035a. Epub 2011 May 9.

Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay

Jennifer Helen McKenzie  1 , K Udeni Alwis, Joanne E Sordillo, Kesava Srinivas Kalluri, Donald Kirby Milton
Affiliations

Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay

Jennifer Helen McKenzie et al. J Environ Monit. 2011 Jun.

Abstract

Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology.

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Figures

Fig. 1
Fig. 1
Summary of experimental design and methods.
Fig. 2
Fig. 2
Effect of assay media on mean endotoxin levels reported by recombinant Factor C assay. Mean endotoxin levels (95% CI) of dust samples (n=60) were determined by rFC assay using Tris buffer, TAP buffer, Tween or water across three lots of rFC assay kits. Differences in mean endotoxin levels were significant (p < 0.001).
Fig. 3
Fig. 3
Effect of lot by assay media type on mean endotoxin levels reported by recombinant Factor C assay. Mean endotoxin levels (95% CI) of dust samples (n=20) reported by each of three lots of rFC assay performed in Tris buffer (A), TAP buffer (B), Tween (C) or water (D).
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