Characterization of the allergen Der f 7 from house dust mite extracts by species-specific and crossreactive monoclonal antibodies
Article first published online: 28 JUN 2008
Clinical & Experimental Allergy
Volume 27, Issue 7, pages 824–832, July 1997
How to Cite
SHEN, H.-D., LIN, W.-L., TSAI, L.-C., TAM, M. F., CHUA, K.-Y., CHEN, H.-L., HSIEH, K.-H., LI, C.-S. and THOMAS, W. R. (1997), Characterization of the allergen Der f 7 from house dust mite extracts by species-specific and crossreactive monoclonal antibodies. Clinical & Experimental Allergy, 27: 824–832. doi: 10.1046/j.1365-2222.1997.650890.x
- Issue published online: 28 JUN 2008
- Article first published online: 28 JUN 2008
- Submitted 22 August 1996; revised 21 November 1996; accepted 24 December 1996.
- mite allergen;
- Der f 7;
- monoclonal antibody;
- IgE binding;
- two-site ELISA
Background The group 7 mite allergens react with IgE in 50% of sera from allergic patients.
Objective To determine the molecular and antigenic characteristics and heterogeneity of Der f 7 in mite extracts.
Methods Monoclonal antibodies (MoAbs) produced from mice immunized with recombinant Der f 7 were examined for crossreactivity to Der p 7 and then used for immunoblotting of 1 and 2-D gel electrophoresis. Deglycosylation was studied with N-glycosidase-F and N-terminal sequencing by Edman degradation. The epitopes of the monoclonal antibodies were compared by cross-inhibitory immunoassays.
Results Immunoblotting of D. farinae extracts with all the anti Der f 7 MoAbs showed major reactivities at 31, 30 and 25 kDa. The strongest immunostaining was at 25 kDa which contrasted with Der p 7 where the 31 and 30 kDa bands were strongest. The relative strength of staining however varied between extracts. The 31 and 30 kDa components were glycosylation products of the 25 kDa form which had the N-terminal sequence predicted from cDNA analysis. Two MoAbs stained an 18 kDa band consistent with a degradation product. The 2-D gels showed that different components with pls from 5.6–6.4. Both species-specific and Der p 7 crossreactive MoAbs were produced and a two-site ELISA assay for detecting group 7 allergen was developed with MoAbs recognizing different epitopes.
Conclusions Der f 7 has been defined by its natural N-terminal sequence and MoAbs. It apparently exists as different glycosylation and degradation products in mite extracts, the relative abundance of which differs with different preparations. A two-site ELISA to measure the allergen was developed.