(133)-␤-D-Glucans are carbohydrate polymers found in the cell walls of most fungi, some bacteria, yeasts, and higher and lower plants. The molecular weight, degree of (136)-linked side branching, helical conformation, and solubility are the most important factors which determine the biological activities of (133)-␤-D-glucans. The biological activities of (133)-␤-D-glucans include host-mediated antitumor activity (11), adjuvant effects (21), and activation of neutrophils (34), eosinophils (23), macrophages (15, 27), and complement (26).There is increasing evidence that (133)-␤-D-glucans cause nonspecific inflammatory reactions (9, 10) and are responsible for bioaerosol-induced respiratory symptoms observed in both indoor (24,25,32) and occupational (4, 5) environments. Dose-response relationships have been found between levels of (133)-␤-D-glucans and eye and throat irritation, dry cough, and itching skin (25). Relationships have also been found among exposure to (133)-␤-D-glucans and an increased prevalence of atopy, a decrease in forced expiratory volume in 1 s in adults (30), and peak flow variability in children (6). Recent studies have reported significantly high levels of (133) (18,20).In the present study, we describe the use of an enzymelinked immunosorbent assay (ELISA), which was originally developed as a diagnostic assay for systemic fungemia (8), to assay (133)-␤-D-glucans in house dust samples, some mould cultures, and metal working fluid samples. The assay is based on the use of a high-affinity receptor (galactosyl ceramide, a sphingolipid) specific for (133)-␤-D-glucans as a capture reagent and a monoclonal antibody specific for complex fungal cell wall (133)-␤-D-glucans as a detector reagent. The assay is reported to be rapid, specific for fungal ␤-glucans, and insensitive to interference caused by agents which affect the Limulus assay. MATERIALS AND METHODSGalactosyl ceramide (Galactercerebroside type II; Sigma, St. Louis, Mo.) and 1% bovine serum albumin (BSA) (Sigma) in Tris-buffered saline (pH 8.0) (TBS) were used, respectively, for coating and blocking. Immulon 96-well polystyrene

LAL and rFC gave comparable results, hence either assay can be used for studies of endotoxin exposure. The current study shows that (1 --> 3)-beta-D-glucan is not a major factor interfering with endotoxin measurements in house dust using a Cambrex KQCL LAL preparation.

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.

The modifications we made included: use of 3-hydroxy C11:0 and C13:0 methyl esters as internal standards, subjecting calibration standards to phase separation and SPE; addition of water to phase separation; addition of 1-pentadecanol as a carrier; injecting 1:4 diluted TMS derivatives of 3-OHFAs; and monitoring both m/z 131 and 133 ions to improve stability of area measurements for product ions. This method of optimization establishes an appropriate technique for quantification of 3-OHFAs in house dust.