The Importance of Antibody Cross-Reactivity for the Development of Environmental Immunoassays for Alternaria Alternata

Abstract

Alternaria alternata is one of the most common fungi indoors and outdoors and exposure to the fungus has been linked to the severity of asthma and the pathophysiology of chronic rhinosinusitis. A recent national survey of house dust samples concluded that 95-99% of American homes contained detectable amounts of Alternaria antigens when analyzed with a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies (pAbs) can lack assay specificity and in this study we investigated the cross-reactivity of the pAbs to other common fungal contaminants. Reactivity to 24 fungal species typically found in indoor environments was analyzed by inhibition ELISA. The pAbs were also tested by immunoblotting and Halogen immunoassay (HIA) for a subgroup of fungi. Spores of 6 fungi including species of Alternaria, Ulocladium, Stemphylium, Epicoccum, Drechslera and Exserohilum were found to react with the pAbs more strongly than A. alternata when tested by ELISA. Six other fungi reacted in the ELISA at a lower level and 11 fungal species including several Penicillium, Aspergillus, Fusarium and Cladosporium species were not found to inhibit pAb binding. The immunoblots and the HIA staining confirmed that the pAbs react with numerous fungi commonly found in US homes. Due to extensive cross-reactivity, the ELISA prevalence data previously reported for A. alternata should be considered to represent fungal-specific antigens rather than be interpreted as exclusive contamination with A. alternata alone. The characterization of antibody specificity before application is critical to avoid analytical bias and ambiguities in data interpretation.