Limitations of monoclonal antibodies for monitoring of fungal aerosols using Penicillium brevicompactum as a model fungus.

Molds are ubiquitous in every environment and many species have been recently associated with an increase in opportunistic infections in immunocompromised patients or the exacerbation of asthmatic episodes in allergic patients. The degree of environmental contamination with fungi thus needs to be monitored and in this study we report the development of a monoclonal antibody (mAb)-mediated enzyme-linked immunosorbent assay (ELISA) for the detection of spores of Penicillium brevicompactum in experimental model aerosols. In addition, we have investigated the influence of different parameters of air sampling and sample recovery on ELISA performance. MAbs were produced with standard hybridoma techniques and cross-reactivities were determined against spores of 53 fungal species by indirect ELISA. Standardized experimental fungal aerosols were collected with the Button Personal Inhalable Aerosol Sampler onto polycarbonate or polytetrafluoroethylene filters (PTFE) and the effects of different extraction buffers and filter agitation methods during sample processing on spore recovery and ELISA detection were investigated. Five mAbs were produced and all of them cross-reacted with several of 31 related Aspergillus, Penicillium and Eurotium species. However, cross-reactivities with 21 non-related fungi were rare. Spores were recovered in much higher numbers from polycarbonate filters (PFs) than from polytetrafluoroethylene filters. Optical densities (ODs) in ELISA were higher for spores collected into carbonate coating buffer (CCB) than phosphate-buffered saline (PBS). Filter bath sonication following filter vortexing had no positive effects on ELISA sensitivity. The cross-reactivity patterns of mAbs suggest that Aspergillus and Penicillium species share multiple antigens. Quantitative ELISA results for fungal aerosols were found to be influenced by differential sample processing and thus method standardization will be essential to maintain the comparability of immunometric monitoring results.