Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3) Horng-Der Shen, PhDa, Chih-Wen Wang, BSca,

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Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3) Horng-Der Shen, PhDa, Chih-Wen Wang, BSca, Hong Chou, PhDa, Win-Ling Lin, BSca, Ming F. Tam, PhDb, Mei-Hsiang Huang, BSca, Ming-Ling Kuo, PhDc, Soo-Ray Wang, MD, PhDd, Shou-Hwa Han, MD, PhDe Journal of Allergy and Clinical Immunology Volume 105, Issue 4, Pages 827-833 (April 2000) DOI: 10.1067/mai.2000.105220 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Nucleotide and inferred amino acid sequences of cDNA clone PCE2. Numbers to the right are nucleotide positions. Putative glycosylation site (Asn-Ala-Ser) and TAA stop codon are underlined. Journal of Allergy and Clinical Immunology 2000 105, 827-833DOI: (10.1067/mai.2000.105220) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 Comparison of deduced amino acid sequence of PCE2 and peroxisomal membrane protein allergen (Asp f 3) of A fumigatus. Residues identical to PCE2 sequence are indicated by dot. Dashes, Spaces that have been introduced to optimize alignment. Putative N-glycosylation site is underlined. Journal of Allergy and Clinical Immunology 2000 105, 827-833DOI: (10.1067/mai.2000.105220) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 IgE immunoblot reactivity to components of P citrinum in asthmatic serum samples. A, Coomassie blue–stained protein profile of P citrinum extract on PVDF membrane. Immunoblots generated from 29 asthmatic serum samples are presented in B. Sera 16, 18, 19, and 20 have negative IgE immunoblotting. Serum 29 was a negative control obtained from mite-sensitive asthmatic patient. Molecular mass standards (Pharmacia, Uppsala, Sweden) used were phosphorylase b (94 kd), BSA (67 kd), ovalbumin (43 kd), carbonic anhydrase (30 kd), soybean trypsin inhibitor (20.1 kd), and α-lactalbumin (14.4 kd). Results of commercial Pharmacia CAP test to P notatum of individual serum sample tested are expressed as RAST classes and are shown in B. Journal of Allergy and Clinical Immunology 2000 105, 827-833DOI: (10.1067/mai.2000.105220) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Immunoblot reactivity of PCE2-GST and Asp f 3-GST fusion proteins to IgE antibodies in asthmatic serum samples. A, Coomassie blue–stained SDS-PAGE protein profiles of affinity-purified PCE2-GST and Asp f 3–GST fusion proteins. Strips in B show immunoblot reactivity of recombinant proteins to IgE antibodies in sera from 13 asthmatic patients (serum No. 1-13 in Fig 3). Strip No. 14 represents result of cord serum sample. Journal of Allergy and Clinical Immunology 2000 105, 827-833DOI: (10.1067/mai.2000.105220) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 Inhibition of IgE immunoblot reactivity of a mixture of serum samples (serum No. 1, 3, 5, 7, 8, 11, and 12 in Figs 3 and 4) that has positive reactivity to PCE2 (A) and Asp f 3 (B). Strips 1 in both panels show Coomassie blue–stained protein profiles of recombinant PCE2 and Asp f 3 on PVDF membranes. Strips 2 show IgE immunoblot reactivity of serum mixture against individual recombinant allergen. Strips 3 to 8 represent results of immunoblot obtained by incubating PVDF blots with same serum mixture that has been mixed and preincubated with 1 and 5 μg, respectively, of PCE2 (strips 3 and 4), Asp f 3 (strips 5 and 6), and BSA (strips 7 and 8). Journal of Allergy and Clinical Immunology 2000 105, 827-833DOI: (10.1067/mai.2000.105220) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 6 Immunoblot reactivity of 6 mAbs to recombinant PCE2 (I-B), Asp f 3 (I-D), crude fungal extracts of P citrinum(II-B) and A fumigatus(II-D). I-A, I-C, II-A, and II-C, Coomassie blue–stained SDS-PAGE protein profiles of the PCE2-GST, Asp f 3–GST, P citrinum, and A fumigatus fungal extracts, respectively. Strips 1-6 in B and D summarize immunoblot reactivity of mAbs PCE2-3, PCE2-7, PCE2-11, PCE2-15, PCE2-19, and PCE2-21 to recombinant proteins (I) and crude fungal extracts (II), respectively. Culture supernatant containing mAb WH20 against house dust mite allergen Der p 7 shows negative result (strips 7). Strips 8 and 9 in all panels show immunoblot results with serum No. 8 in Figs 3 and 4 and a cord serum sample, respectively. Journal of Allergy and Clinical Immunology 2000 105, 827-833DOI: (10.1067/mai.2000.105220) Copyright © 2000 Mosby, Inc. Terms and Conditions