title: Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay.
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.
authors.affiliation: Biomedical Engineering and Biotechnology Program, University of Massachusetts, Lowell, MA, USA.
authors.forename: Jennifer Helen
authors.initials: JH
authors.lastname: McKenzie
authors.forename: K Udeni
authors.initials: KU
authors.lastname: Alwis
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Sordillo
authors.forename: Kesava Srinivas
authors.initials: KS
authors.lastname: Kalluri
authors.forename: Donald Kirby
authors.initials: DK
authors.lastname: Milton
mesh_terms: D000818:Animals; D060829:Arthropod Proteins; D001681:Biological Assay / Q000379:methods*; D004731:Endotoxins / Q000032:analysis*; D004784:Environmental Monitoring / Q000379:methods*; D004785:Environmental Pollutants / Q000032:analysis*; D004792:Enzyme Precursors; D005227:Fatty Acids / Q000032:analysis; D006737:Horseshoe Crabs; D015203:Reproducibility of Results; D012697:Serine Endopeptidases
chemical_list: D060829:Arthropod Proteins; D004731:Endotoxins; D004785:Environmental Pollutants; D004792:Enzyme Precursors; D005227:Fatty Acids; D012697:Serine Endopeptidases; C057151:Limulus clotting factor C