Detection of Fungal Cell Wall beta-Glucans in Murine and Human Biological Fluids Using a Sensitive Fungal beta-Glucan-Specific Capture ELISA.



Fungal cell wall beta-glucans are shed by actively growing fungi and are detectable in plasma samples from patients with systemic candidemia or aspergillomycosis. Current beta-glucan detection techniques are largely based on the Limulus coagulation cascade and are problematic due to assay interferences by endotoxin, other bacterial components, non-fungal carbohydrate polymers and by natural biological variability of Limulus lysates. We have developed a novel capture ELISA that sensitively detects beta-glucans from Candida, Aspergillus and other fungi in a variety of complex biological fluids, including plasma and urine. The ELISA utilizes a high-affinity beta-glucan receptor purified from human leukocytes as a capture reagent and a monoclonal antibody specific for complex fungal cell wall beta-glucans as the detection reagent. The capture ELISA is rapid and specific for beta-glucans with no detectable interference by bacterial components or non-fungal carbohydrate polymers. Experiments in which mouse and human plasma or urine samples were spiked with known concentrations of beta-glucans demonstrated that the level of fungal beta-glucan detection in urine is 300 pg/mL and 1 ng/mL in plasma. Plasma, urine, and kidney tissue samples were collected from mice experimentally infected with Candida, Aspergillus at various time points post-infection and analyzed for the presence of beta-glucans using the ELISA or for positive microbiological cultures. beta-glucans were readily detected in each of the samples taken from infected animals 24 hours post-infection even though microbiological cultures were negative. Samples collected from uninfected, control animals had no detectable beta-glucans or positive cultures at this time point. These data support the use of fungal beta-glucans as a diagnostic marker of systemic candidemia and aspergillomycoses and illustrate the potential utility of the beta-glucan capture ELISA in the early detection of fungal infections.

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38th Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 38th