CHARACTERIZATION OF THE ALLERGEN DER-F-7 FROM HOUSE-DUST MITE EXTRACTS BY SPECIES-SPECIFIC AND CROSS-REACTIVE MONOCLONAL-ANTIBODIES

Background The group 7 mite allergens react with IgE in 50% of sera fr om allergic patients. Objective To determine the molecular and antigen ic characteristics and heterogeneity of Der f 7 in mite extracts. Meth ods Monoclonal antibodies (Moabs) produced from mice immunized with re combinant Der f 7 were examined for crossreactivity to Der p 7 and the n used for immunoblotting of 1 and 2-D gel electrophoresis. Deglycosyl ation was studied with N-glycosidase-F and N-terminal sequencing by Ed man degradation. The epitopes of the monoclonal antibodies were compar ed by cross-inhibitory immunoassays.Results Immunoblotting of D. farin ae 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. Th e 31 and 30 kDA components were glycosylation products of the 25 kDa f orm which had the N-terminal sequence predicted from cDNA analysis. Tw o 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. B oth species-specific and Der p 7 crossreactive MoAbs were produced and a two-site ELISA assay for detecting group 7 allergen was developed w ith MoAbs recognizing different epitopes. Conclusions Der f 7 has been defined by its natural N-terminal sequence and MoAbs. It apparently e xists as different glycosylation and degradation products in mite extr acts, the relative abundance of which differs with different preparati ons. A two-site ELISA to measure the allergen was developed.