Analytical bias of cross-reactive polyclonal antibodies for environmental immunoassays of Alternaria alternata

Abstract

Alternaria alternata is recognized as an important aeroallergen indoors and outdoors, and exposure to the fungus has been identified as a risk factor for asthma. Two recent publications concluded that 95% to 99% of American homes contained detectable amounts of Alternaria antigens when analyzed with a polyclonal antibody (pAb)-based ELISA. We investigated the cross-reactivity of the commercially available pAbs that were used in those studies. Reactivity to 24 fungal species commonly found in indoor environments was analyzed by inhibition ELISA by using solid-phase A alternata antigen. The pAbs were also tested by immunoblotting and halogen immunoassay for a subgroup of fungi. Spores of 7 fungi including species of Alternaria, Ulocladium, Stemphylium, Epicoccum, Drechslera, and Exserohilum strongly inhibited the binding of the pAbs 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 failed to show inhibition. The immunoblots and the halogen immunoassay staining confirmed the cross-reactivity patterns of the ELISA. The pAbs against A alternata were found to cross-react broadly with related and nonrelated fungi. The prevalence data previously reported for A alternata should be considered to be fungal-reactive rather than A alternata-specific.