Identification, characterization, and cloning of a complementary DNA encoding a 60-kd house dust mite allergen (Der f 18) for human beings and dogs Eric.

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Identification, characterization, and cloning of a complementary DNA encoding a 60-kd house dust mite allergen (Der f 18) for human beings and dogs Eric Weber, PhDa*, Shirley Hunter, PhDa, Kim Stedman, BSa, Steve Dreitz, BSa, Thierry Olivry, DrVet, PhDb, Andrew Hillier, BVScc, Catherine McCall, DPhila Journal of Allergy and Clinical Immunology Volume 112, Issue 1, Pages 79-86 (July 2003) DOI: 10.1067/mai.2003.1602 Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 1 A, Coomassie-stained PAGE of purified Der f 18. Two micrograms of purified Der f 18 was resolved on a 14% SDS reducing PAGE gel, stained with Coomassie Blue, and destained. B, Canine IgE Western blot of purified native Der f 18. Approximately 5 ng of purified Der f 18 was resolved on a 14% SDS reducing PAGE gel. Journal of Allergy and Clinical Immunology 2003 112, 79-86DOI: (10.1067/mai.2003.1602) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 2 Nucleotide sequence of Der f 18 cDNA with deduced amino acid sequence. From top to bottom , the ATG initiation codon, amino acids encoding the secretory leader peptide, N-glycosylation site (N-I-T, aa 338-340), stop codon (TGA), and the putative polyadenylation signal (AATAAA) are in bold . Internal peptide sequences generated by protein sequencing are underlined . Journal of Allergy and Clinical Immunology 2003 112, 79-86DOI: (10.1067/mai.2003.1602) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 3 Regional protein sequence alignment of Der f 18 with homologues showing conserved cysteine residues, labeled C1 to C4. The conserved residues within the 2 catalytic domains are labeled CD1 and CD2, respectively. Journal of Allergy and Clinical Immunology 2003 112, 79-86DOI: (10.1067/mai.2003.1602) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 4 Immunostaining of Der f 18 in whole body sections of D farinae . A, Paraffin-embedded sections were immunostained with mAb H407 specific for Der f 18 by using a 3-step streptavidin-peroxidase method. This putative chitinase was localized to the upper digestive tract of the mites, such as the esophagus (as seen in the longitudinal section at left ), or the ventriculus (as seen in the longitudinal, sagittal, and cross-sections of the mites). Immunostaining of the mites was carried out with an irrelevant mAb (B) . Of note is that in all longitudinal and sagittal mite sections, the cuticle covering the cranial body and proximal front exhibited a brown color. This color is considered normal for D farinae house dust mites. Bar = 60 μm. Journal of Allergy and Clinical Immunology 2003 112, 79-86DOI: (10.1067/mai.2003.1602) Copyright © 2003 Mosby, Inc. Terms and Conditions