A new immunoassay to quantify fungal antigens from the indoor mould Aspergillus versicolor Eva Zahradnik, * a Sabine Kespohl, a Ingrid Sander, a Ursula Schies, b Janett Khosravie- Hohn, b Wolfgang Lorenz, c Steffen Engelhart, d Annette Kolk, e Gerd Schneider, e Thomas Br¨ uning a and Monika Raulf-Heimsoth a Aspergillus versicolor is among the most commonly found moulds in moisture-damaged buildings and can be associated with adverse health effects in humans. This paper reports the development, validation and application of an enzyme immunoassay to quantify A. versicolor antigens. A sandwich ELISA was developed using polyclonal antibodies that recognize a broad range of A. versicolor proteins present in fungal spores and in mycelia fragments. To validate the new method, A. versicolor antigens were quantified in samples collected from homes with visible mould growth, including dust from vacuumed walls and bulk samples of building materials. Antigen concentrations were compared to the results of a commercial ELISA based on monoclonal antibodies (AveX ELISA, Indoor Biotechnologies, Charlottesville, USA) and correlated with colony forming units (CFU) of A. versicolor. The A. versicolor ELISA was very sensitive with a lower detection limit of 120 pg ml 1 . The assay also showed some reactivity to other moulds with strongest reactions with other Aspergillus species (1–3% reactivity). The new assay detected A. versicolor antigens in a much higher percentage of dust samples (88% vs. 27%) and bulk samples (89% vs. 24%) than the AveX assay. A significant correlation (r ¼ 0.67, and p < 0.0001) was found between antigen concentrations and CFU of A. versicolor. Based on its low detection limit and good correlation with the culture-based method, this new immunoassay seems to be a useful tool for the measurement of A. versicolor exposure levels and a reliable complement to the traditional monitoring techniques, such as mould cultivation or microscopy. Environmental impact Dampness and increased mould growth in the indoor environment are associated with allergies and respiratory diseases. Classical methods to assess fungal exposure comprise the determination of viable moulds by the counting of colony forming units. The viability of the mould is not essential to induce immu- nologic health effects. Therefore, mould antigen concentrations may be a more suitable measure for exposure assessment than culturable fungi. This paper reports the development, validation and application of an enzyme immunoassay to quantify the indoor mould Aspergillus versicolor which has been classied as a moisture indicator. With the sandwich ELISA described in the manuscript, it is possible to detect a broad range of different A. versicolor antigens existing in spores and mycelia. Based on the low detection limit, sufficient specicity and good correlation with the colony forming units, the new immunoassay can be a useful tool to monitor antigen levels of A. versicolor in occupational and domestic environments. Introduction Exposure to moulds may induce various human disorders, such as infections, irritations and allergic reactions. Fungal exposure can also increase asthma symptoms, prevalence and severity. 1,2 The fungi most commonly recovered in different regions from both indoor and outdoor air are Cladosporium, Penicillium, Aspergillus and Alternaria in the descending order. 3–6 Dampness or water damage in homes is oen associated with increased mould growth, possibly resulting in health problems of the occupants. 7,8 Aspergillus versicolor is one of the most common fungi found in different materials and air samples from mois- ture-damaged buildings. 9–12 Besides its properties as a moisture indicator, A. versicolor produces harmful mycotoxins, particu- larly the carcinogenic sterigmatocystin. 13,14 Using human sera of mould-sensitized patients, several allergens have been identi- ed in protein extracts from A. versicolor spores. 15,16 One such allergen, an alkaline serine protease, was approved by the IUIS a Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-Universit¨ at Bochum (IPA), Center of Allergology/Immunology, Buerkle-de-la-Camp-Platz 1, 44789, Bochum, Germany. E-mail: zahradnik@ipa-dguv.de b German Social Accident Insurance Institution for Trade and Distribution Industry, Munich, Germany c Dr Lorenz Institut f¨ ur Innenraumdiagnostik, D¨ usseldorf, Germany d Institute for Hygiene and Public Health, University of Bonn, Bonn, Germany e Institute for Occupational Safety and Health of the German Social Accident Insurance (IFA), Sankt Augustin, Germany Cite this: Environ. Sci.: Processes Impacts, 2013, 15, 1162 Received 29th October 2012 Accepted 16th April 2013 DOI: 10.1039/c3em30870g rsc.li/process-impacts 1162 | Environ. Sci.: Processes Impacts, 2013, 15, 1162–1171 This journal is ª The Royal Society of Chemistry 2013 Environmental Science Processes & Impacts PAPER