Isolation and characterization of a novel 98-kd Dermatophagoides farinae mite allergen  Lai-Chen Tsai, BSca b, Pei-Ling Chao, MSa, Horng-Der Shen, PhDa b, Ren-Bin Tang, MDc, Tsu-Chung Chang, PhDd, Zo-Nan Chang, BSce, Mei-Whey Hung, BSca, Bo-Liang Lee, MSa, Kaw-Yan Chua, PhDf  Journal of Allergy and Clinical Immunology  Volume 102, Issue 2, Pages 295-303 (August 1998) DOI: 10.1016/S0091-6749(98)70099-5 Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 1 SDS-PAGE and immunoblotting profile of crude Df mite extract and purified Df642 protein. Proteins were separated under reducing condition on 10% SDS-polyacrylamide gel. A, Separated proteins of whole mite extract (5 μg, lane 1) and Df642 (1 μg, lane 2) were visualized by staining with Coomassie brilliant blue. Protein profile of Df642 component with an MW of about 98 kd was abundant. B, Separated proteins of mite extract (lane 1) and fresh preparative Df642 (lanes 2 to 4) were electrotransferred to PVDF membrane and immunostained by probing with mAb642 (lanes 1 and 2), mAb11 (lane 3), and mAb412 (lane 4). The 98-kd band reacted specifically with mAb Df642 (lane 2) but not with mAb11 or mAb412 (lanes 3 and 4). Protein profile of Df crude extract (lane 1) was similar to that of Df642, but reactivity was much reduced. Journal of Allergy and Clinical Immunology 1998 102, 295-303DOI: (10.1016/S0091-6749(98)70099-5) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 2 2D-immunoblotting activity of mAb642 to crude extract of Df mite (A and B) and purified Df642 (C). Protein profile of whole mite extract with silver staining after SDS-PAGE (A) and reactivity of mAb642 to mite extract by immunoblot (B). Df642 protein profile with silver staining (C 2) was similar on probing with mAb642 (C 3). Arrows in A and C 2 indicate protein spots corresponding to those that showed enzyme color reaction in immunoblotting as shown in B and C 3. The isoelectric point standard is shown in A and C 1. Journal of Allergy and Clinical Immunology 1998 102, 295-303DOI: (10.1016/S0091-6749(98)70099-5) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 3 IgE reactivity to purified Df642 by immunoblotting. Mite extract (10 μg/lane; lanes 1 to 3), Df642 (1 μg/lane; lanes 4 to 6), Aspergillus fumigatus extract (10 μg/lane; lane 7), GST-Dp2 fusion protein (1 μg/lane; lanes 8 and 9), and GST (1 μg/lane; lane 10) were separated on 10% SDS-polyacrylamide gel and electrotransferred to PVDF membrane. The membrane strips were probed with the nonabsorbed serum from patients allergic to mites (lanes 1, 4, 8 and 10) and with same serum preabsorbed with 25 μg (lane 2) and 50 μg (lanes 3 and 9) Df642. Other strips were probed with control nonallergic serum (lane 5) and A. fumigatus–allergic serum (lanes 6 and 7). Journal of Allergy and Clinical Immunology 1998 102, 295-303DOI: (10.1016/S0091-6749(98)70099-5) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 4 IgE reactivity to purified Df642 by immunodotting. IgE reactivity to Df642 of selected panel of sera from asthmatic patients with positive skin test reactions to mites (0.2 μg/dot) was detected by immunodot. Major mite allergen, Der p 2 (0.2 μg/dot), and Df mite extract (1 μg/dot) were used as positive controls, whereas GST (0.2 μg/dot) was used as negative control. Reactivity was considered positive if intensity of the tested sera were higher than cut-off intensity value (113; mean + 2 SD) from negative control. Intensity value was measured by densitometer and imaging analyzer. Journal of Allergy and Clinical Immunology 1998 102, 295-303DOI: (10.1016/S0091-6749(98)70099-5) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 5 Lectin-binding reactivity of Df642. The binding of lectins to DF642 was detected by glycan differentiation kit. Glycoproteins (0.5 to 1 μg) of carboxypeptidase (lane 1), transferrin (lane 3), fetuin (lane 5), and asialofetuin (desialylated fetuin) (lane 7) were immunoblotted onto PVDF membrane and used as controls for GNA, SNA, MAA, AND PNA, respectively. Lanes 2, 4, 6 and 8 were all immunoblotted with purified Df642 protein (1 μg/stripe). Immunoblotting stripes 1 and 2, 3 and 4, 5 and 6, and 7 and 8 were separately incubated for 1 hour with diluted lectin solutions of digoxigenin-labeled GNA, SNA, MAA, AND PNA, respectively, and then reacted with alkaline phosphatase–conjugated anti-digoxigenin for another 1 hour at room temperature. After washing, the filters were immersed in NBT solution until black color developed. Results indicate that only digoxigenin-labeled GNA reacted with Df642 protein (lane 2.) Journal of Allergy and Clinical Immunology 1998 102, 295-303DOI: (10.1016/S0091-6749(98)70099-5) Copyright © 1998 Mosby, Inc. Terms and Conditions

Fig. 6 Amino acid sequence homology between Df642 and paramyosin from variety of invertebrate species. Comparison of composite alignments of deduced internal amino acid sequence of Df642 (row a) with muscle paramyosin from Drosophila melanogaster (row b), Caenorhabditis elegans (row c), Dirofilaria immitis (row d), and Onchocerca volvulus (row e). Sequences are shown by 1-letter code. Asterisks indicate position in alignment is perfectly conserved; open circles indicate position is well conserved. Journal of Allergy and Clinical Immunology 1998 102, 295-303DOI: (10.1016/S0091-6749(98)70099-5) Copyright © 1998 Mosby, Inc. Terms and Conditions