For the first time the complete cDNA encoding a major allergen and novel protein of the yeast Malassezia furfur, Ma1 f 1, has been sequenced and expressed. The amino acid sequences of nine tryptic peptides of the protein were determined. Oligonucleotides were designed from these amino acid sequences. The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by reverse-transcnptase PCR techniques. [I, 21. However, under the influence of predisposing factors, this yeast may induce allergic reactions in atopic dermatitis. The importance of M. furjiur in the pathogenesis of this disease has been investigated during the last 1 0 years and, in 40-65 % of patients, activity against M. furjiur has been shown with skin tests and/or specific IgE serology [3-81.Numerous IgE-binding components with molecular masses between 10-100 kDa have been identified by immunochemical methods [9-121. We have previously reported the production of a specific mouse monoclonal antibody (mAb 9G9) against one major allergen of M. furjiur of approximately 37 kDa [9]. This protein is now designated as Ma1 f 1 according to the new allergen nomenclature [13]. This work reports the complete cDNA sequence of a novel protein from the yeast M. furjiur that shows the criteria of a major allergen in atopic dermatitis. Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and is available under accession number X96486. 4 days at 37°C. The yeast extract was prepared as described earlier [9]. The proteins were reduced, alkylated with 50 mM iodoacetamide and separated on a 12.5 % SDS/polyacrylamide gel. Proteins were visualized by Coomassie blue staining and the allergen verified by immunoblotting using our mAb 9G9. MATERIALS AND METHODS IsolationProteolytic digestion, fractionation of internal fragments. The stained protein band was excised from the gel and subjected to in-gel digestion followed by separation of peptides [14]. Briefly, the gel piece was washed, dried and digested with 0.5 pg modified trypsin, sequence grade (Promega, Scandinavian Diagnostic Services) at 30°C overnight. The generated fragments were extracted and isolated by narrow-bore reverse-phase liquid chromatography using a column of pRPC C2/C18 SC 2.1/10 operated in a SMART system (Pharmacia Biotech). A linear gradient of acetonitrile (0.25 %/min) in 0.06 '% trifluoroacetic acid was used at a flow rate of 100 pl/min to elute the peptides. The eluate was monitored at 215 nm and 280 nm.ELISA assay. 54 fractions of the tryptic protein digest were analyzed by the standard ELlSA technique using our specific mAb 9G9 [9]. Briefly, the peptides were coated onto an ELISA plate, blocked with phosphate-buffered saline (137 mM NaCI, 2.7 mM KCI, 10.5 mM NaHPO,, 1.7 mM KH,PO,, pH 7.4) containing 0.5 % BSA and incubated overnight at room temperature with the mAb 9G9. After washing with 0.9% NaCI/O.S% Tween 20, the wells were incubated for 4 h at room temperature with alkaline-phosphatase-conjugated rabbit anti-mouse ...

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.

Our data suggest that P. orbiculare can induce a T-cell response in AD patients. The Th2-like profile of P. orbiculare-reactive TCC derived from lesional skin indicates that P. orbiculare may play a role in maintaining IgE-mediated skin inflammation in AD.

Allergen-specific IL-4 producing cells in a peripheral blood mononuclear cell (PBMC) culture can be detected by ELISPOT and the response can synergistically be enhanced by suboptimal concentrations of PHA.

We have developed a sensitive, horse-allergen-specific, mAb assay allowing detection of low levels of horse allergens. Raised levels of horse allergen were found outdoors only in the close vicinity of the stable.