Quantification of airborne fungal antigens by ELISA and comparison to molecular biological and classical methods

Airborne fungal spores are recognized for their human health impact, yet dose-response relationships remain undefined despite decades of bioaerosol sampling and analysis. In this article, we quantify immunologically active compounds, using six fungal-specific enzyme-linked immunosorbent assays (ELISAs) and compare them with cultivation, spore counting, and quantitative PCR (qPCR), using identical filter extract aliquots, various concentrations, single-organism aerosols, and mixes, in a controlled environment. Results showed high agreement between ELISAs and spore counting. By contrast, CFU enumeration and qPCR showed lower correlation with the other methods. Results of ELISAs showed high reproducibility of technical replicates and sampling duplicates, whereas CFU and qPCR results had high deviations. ELISA detection limits ranged from 10 to 100 spores/mL for larger spores (Cladosporium herbarum, Aspergillus amstelodami) and from 103 to 104 spores/mL for smaller spores (Aspergillus amoenus, Aspergillus fumigatus, Penicillium chrysogenum). The Wallemia sebi and the Penicillium chrysogenum ELISAs cross-reacted to Aspergillus protuberus or Aspergillus amstelodami, respectively. The spore dust production method influenced the spore germination rate and qPCR results significantly. By contrast, ELISA results remain unchanged, with the exception of the W. sebi ELISA. Quantitative spore counting was challenged by mixtures of species with morphologically similar spores from different fungal groups. In conclusion, the ELISA method is found suitable for fungal antigen quantification of air samples, provided the assays are sufficiently sensitive and specific, and germination evaluation is not required for risk assessment, leading to C. herbarum and A. amstelodami ELISA recommendations.

Bioaerosol detection and analysis is an ongoing field of research. Although various methodologies are used for collection and analysis, there is no single method available to close the knowledge gap between exposure and health impact. For airborne fungal material, the standard analysis method remains cultivation. With molecular technology advancing in the field, both methods can only show the exposure to living, cultivatable, or total fungal cells in the airborne environment. To close the gap between airborne concentrations and impact on the human body, recognition of the allergenic potential is necessary. Therefore, we evaluated six fungal-specific ELISAs to make them ready for application in field studies and compared them to cultivation, spore counting, and molecular genetic methods. We are confident that in the future antigen-recognizing methods like the tested ELISAs will enable moving from particle detection toward detection of the disease-causing agent.