Halogen Immunoassay, a New Method for the Detection of Sensitization to Fungal Allergens; Comparisons with Conventional Techniques

Abstract

Accurate diagnosis of allergy to specific fungal species is confounded by the variability in allergens occurring with different diagnostic systems. We compared the halogen immunoassay (HIA), which uses allergens expressed by freshly germinated spores that are bound to protein binding membranes (PBM), with the commercial Pharmacia UniCap assay (CAP) and with skin prick tests (SPT). Serum from 60 subjects was used; 30 were SPT positive and sensitized to at least one of Alternaria alternata or Aspergillus fumigatus and the other 30 were SPT negative to these fungi but known to be sensitized to non-fungal allergens. All sera were analyzed by CAP against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens. For HIA, spores from reference cultures belonging to these four species were germinated on PBM, laminated and then probed with each serum. Two independent observers using an ordinal ranking system quantified the intensity and occurrence of the resultant immunoglobulin E (IgE) immunostained haloes around spores and this was statistically compared with the results of the two conventional immunodiagnostic techniques. Germinated conidia of each species expressed detectable allergen in the HIA. The agreement between the ordinal rank scores assigned by the pair of observers was very good (k >or= 0.8) and only differed for A. fumigatus (k = 0.66) . Between 3% and 7% of SPT negative sera was identified by HIA to have specific IgE towards A. fumigatus and A. alternata. For all four species tested there were strong correlations between HIA and CAP (P < 0.0001). However the correlation of both HIA and CAP to SPT was weaker for A. alternata (r(s) = 0.44, P < 0.0153) and absent for A. fumigatus. Overall, the HIA is a new immunodiagnostic technique for the detection of sensitization to fungal allergens that correlates significantly with CAP and to a lesser extent with SPT. This may be due to extract variability and system differences. The significance of this derives from the unique ability of the HIA to measure IgE antibodies to the undegraded allergens that are actively secreted by germinating conidia and hyphae. These are the natural agents of exposure to fungi, and as such, are most likely to be relevant to clinical disease.