title: Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy.
abstract: Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.
authors.affiliation: National Institute of Occupational Health, Department of Chemical and Biological Work Environment, Oslo, Norway.
authors.forename: Komlavi Anani
authors.initials: KA
authors.lastname: Afanou
authors.affiliation: National Institute of Occupational Health, Department of Chemical and Biological Work Environment, Oslo, Norway.
authors.forename: Anne
authors.initials: A
authors.lastname: Straumfors
authors.affiliation: National Institute of Occupational Health, Department of Chemical and Biological Work Environment, Oslo, Norway.
authors.forename: Asbjørn
authors.initials: A
authors.lastname: Skogstad
authors.affiliation: National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, Morgantown, West Virginia, USA.
authors.forename: Ajay P
authors.initials: AP
authors.lastname: Nayak
authors.affiliation: Norwegian Veterinary Institute, Section of Mycology, Oslo, Norway.
authors.forename: Ida
authors.initials: I
authors.lastname: Skaar
authors.affiliation: Norwegian University of Life Science, Institute of Chemistry, Biotechnology and Food Science, Ås, Norway.
authors.forename: Linda
authors.initials: L
authors.lastname: Hjeljord
authors.affiliation: Norwegian University of Life Science, Institute of Chemistry, Biotechnology and Food Science, Ås, Norway.
authors.forename: Arne
authors.initials: A
authors.lastname: Tronsmo
authors.affiliation: National Institute of Occupational Health, Department of Chemical and Biological Work Environment, Oslo, Norway wijnand.eduard@stami.no.
authors.forename: Wijnand
authors.initials: W
authors.lastname: Eduard
authors.affiliation: National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, Morgantown, West Virginia, USA.
authors.forename: Brett James
authors.initials: BJ
authors.lastname: Green
mesh_terms: D001230:Aspergillus / Q000737:chemistry / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure; D007118:Immunoassay / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D010316:Particle Size; D013172:Spores, Fungal / Q000737:chemistry / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure