A new immunoassay to quantify fungal antigens from the indoor mould Aspergillus versicolor

Abstract

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 = 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.

Ex Vivo Cytokine Release and Pattern Recognition Receptor Expression of Subjects Exposed to Dampness: Pilot Study to Assess the Outcome of Mould Exposure to the Innate Immune System

“…18 of the 25 subjects were poised to lay out one EDC in the room with the moisture damage. Extracts of the dusts collected with EDC were prepared by shaking filters for 1 h in 20 mL PBS with 0.05% Tween, followed by collection of the fluid, and centrifugation for 15 min at 3000 g. The resulting supernatant was used for A. versicolor , Penicillium chrysogenum and β-(1,3)-Glukan quantification as described previously [25–27]. In addition, for endotoxin determination extracts without Tween were prepared.…”

Determination of ATP-activity as a useful tool for monitoring microbial load in aqueous humidifier samples

“…Methods without culturing are for example DNA sequencing (Nonnenmann et al, 2010), fluorescence analysis using DAPI staining (Klug and Jäckel, 2012), measurement of endotoxin activity with the LAL test (Gee et al, 2008;Liebers et al, 2009), detection of pyrogenic activity with whole blood assay (Liebers et al, 2010;Punsmann et al, 2013), determination of fungi antigen or ␤-glucan by the ELISA technique (Sander et al, 2008(Sander et al, , 2012Zahradnik et al, 2013), and ATP content via bioluminescence (Hammes et al, 2010;Mueller et al, 2009;Liebers et al, 2012).…”

Biochemical and immunological analysis of mould skin prick test solution: current status of standardization

“…Aspergillus fumigatus‐, A. versicolor‐, C. herbarum‐ and P. chrysogenum ‐specific polyclonal antibodies were produced and purified as described previously . Human serum samples from three to six subjects with individual mould sensitization and suspected allergic symptoms were used for sIgE detection.…”