title: Structure of the major cat allergen Fel d I in different allergen sources: an immunoblotting analysis with monoclonal antibodies against denatured Fel d I and human IgE.
abstract: In this paper we show the reactivity of monoclonal antibodies (mAbs) and human IgE with Fel d I from different allergen sources in reduced SDS-PAGE immunoblots. By SDS-PAGE analysis of affinity-purified 125I-Fel d I, a 14- to 20-kD band was found, which dissociated under reducing conditions into a 4- to 5-kD chain (chain 1) and a 11- to 15-kD chain (chain 2). In initial immunoblotting experiments with mAbs against Fel d I however, only chain 1 was detected, while the mAbs lost activity upon reduction of Fel d I. Therefore mAbs were raised against reduced and alkylated Fel d I. Two of the four mAbs to 'denatured' Fel d I that were obtained did react with chain 2 on an immunoblot under reducing conditions; the other two reacted with chain 1. The mAbs did not react with native Fel d I. With these mAbs and human IgE, differences between allergen source materials in blot patterns of Fel d I were detected. A variable molecular weight for the protein stained with mAb antichain 2 was found, and occasionally the presence of a 12-kD band stained with mAb antichain 1. Human IgE strongly bound to chain 1 of Fel d I, while only 2 out of 6 sera gave a strong reaction with chain 2. The additional 12-kD band was also recognized by human IgE. In a competitive radioimmunoassay with mAb antichain 1, differences in levels of 'denatured' Fel d I between commercial extracts were quantitated. In vitro 'denatured' Fel d I was generated under high pH conditions. The reactivity of human IgE with this 'denatured' Fel d I was demonstrated in indirect RAST experiments with mAb antichain 1. We conclude that mAb antichain 1 recognizes a form of Fel d I that is not detected by mAb antinative Fel d I, but does react with human IgE.
mesh_terms: D000485:Allergens / Q000032:analysis / Q000737:chemistry*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002415:Cats / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006023:Glycoproteins; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D011489:Protein Denaturation / Q000276:immunology*; D011852:Radioallergosorbent Test; D011863:Radioimmunoassay; D013329:Structure-Activity Relationship
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; D007073:Immunoglobulin E; C059296:Fel d 1 protein, Felis domesticus