Identification of allergen components of the opportunistic yeast Pityrosporum orbiculare by monoclonal antibodies

Abstract

The yeast Pityrosporum orbiculare (P. orbiculare) is a member of the normal human cutaneous flora, but it is also associated with several clinical manifestations of the skin. We have previously observed IgE-binding components in P. orbiculare extracts, using sera from patients with atopic dermatitis. In the present study, we raised several monoclonal antibodies (MoAbs) against P. orbiculare to characterize some of its antigens, and used Candida albicans (C. albicans) as a control. We obtained several IgG1 MoAbs which specifically recognized P. orbiculare in ELISA. Two of these were selected for immunoblotting studies on P. orbiculare, and two patterns of reactivity emerged. Firstly, one MoAb showed a distinct band at a molecular mass of 67 kDa. In the second pattern, a sharp band at about 37 kDa appeared. In contrast, the IgM antibodies raised reacted with a 14-kDa component; but they reacted with C. albicans in addition to P. orbiculare. The IgG1 antibodies seemed to react with proteins, as their ability to react in ELISA with extract pretreated with protease was greatly reduced. In contrast, IgM MoAbs were much less affected, suggesting that they recognized nonprotein components. To determine whether these MoAbs-binding components were also recognized by human IgE, we adopted a radioimmunoassay (RIA) using the MoAbs as catcher antibodies. Both the 67-kDa and the 37-kDa components were IgE-binding proteins. P. orbiculare RAST positive sera were scored as positive in the RIA, whereas the control serum was not.

Cross-Reactivity and 1.4-Å Crystal Structure ofMalassezia sympodialisThioredoxin (Mala s 13), a Member of a New Pan-Allergen Family

“…Allergen-specific IgE to extract was determined by ImmunoCAPs m70 (M. sympodialis) using the Pharmacia CAP system (Pharmacia Biotech) according the package insert. Routine skin-prick tests (SPT) and atopy patch tests (APT) were performed with an in-house M. sympodialis (ATCC strain 42132) extract prepared as described previously (22). SPT and APT with Mala s 13 and human Trx were performed as described elsewhere (23).…”

IgE‐reactivity to seven Malassezia species

“…The mixtures were then incubated with the blotted membranes of various Malassezia extracts. Detection of IgE-binding components was performed as described previously (20). The intensity of the bands was recorded in a scanner and the percentage of the inhibition was analysed with the Image Analysis Systems Software, IASS, (Molecular Analyst Software, BIO RAD, CA).…”

Atopic Dermatitis and Fungi

“…Specific antibodies were found especially in patients with AD and less commonly in patients with other atopic manifestations. This was later verified by other groups using various extract from Malassezia (19,29,30,66,69,157,166). In the study by Broberg at al., specific IgE antibodies were found in eight children with AD, five in the age group from 16 to 21 years, and only one patient with rhinitis and/or asthma (19).…”

“…Several IgE binding components of Malassezia have been isolated (29,30,62,66,69,70,98,131,132,137,149,157,166,167). Three major allergen components from Malassezia were later identified using monoclonal antibodies (166).…”