BACKGROUND: The major dog allergens, Can f 1 and Can f 2, are members of the lipocalin protein family. The characterization of both dog allergens is still not complete. Their deduced amino acid sequences indicate the presence of three cysteine residues, probably connected with a disulfide bridge. We compared the biochemical and immunological properties of Can f 1 with those of Can f 2 using gel filtration, electrophoresis, and immunological assays. METHODS: The rCan f 1, rCan f 2 and dog salivary proteins containing natural Can f 1 and Can f 2 were analyzed by HPLC gel filtration. The recombinant Can f 1 (rCan f 1) and rCan f 2 were analyzed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with or without reduction. The binding ability of rabbit IgG purified by protein G affinity chromatography from the antiserum against rCan f 1 and rCan f 2 was examined after a reduction in the recombinant allergens. The immunological cross-reaction between rCan f 1 and rCan f 2 was examined by an enzyme-linked immunosorbent assay (ELISA) using the rabbit IgG against rCan f 1 and rCan f 2. The cross-reaction of human IgE in the serum of a patient with dog allergy between rCan f 1 and rCan f 2 was also analyzed by competitive ELISA. RESULTS: The molecular weights of rCan f 1 and of rCan f 2 were 18 and 21 kDa, respectively, using SDS-PAGE under reducing conditions, but the natural Can f 1 and Can f 2 were separated by HPLC gel filtration into fractions containing proteins of 31 and 34 kDa, respectively. rCan f 1 and rCan f 2 migrated as multiple bands (30-100 kDa) in native PAGE in the presence or absence of a reductant. The molecular weights of natural Can f 1 and of Can f 2 were 20 and 23 kDa, respectively, in SDS-PAGE under reducing conditions. The ability of rabbit IgG to bind to rCan f 1 and rCan f 2 increased after the reduction of the recombinant allergens. The rabbit IgG against rCan f 1 bound to rCan f 2. Cross-reaction of human IgE was observed between rCan f 1 and rCan f 2. CONCLUSIONS: In the native and recombinant forms, Can f 1 and Can f 2 possessed a dimer structure under natural (non-reduced) condition. The dimers of Can f 1 and of Can f 2 were not built with a disulfide bridge but by non-covalent association. Cleavage of a disulfide bond of rCan f 1 and rCan f 2 increased the ability of binding of rabbit IgG to the allergens. The cross-reactivity of rabbit IgG and human IgE between rCan f 1 and rCan f 2 indicates that the same epitope(s) was present in Can f 1 and Can f 2.
BACKGROUND: The major dog allergens, Can f 1 and Can f 2, are members of the lipocalin protein family. The characterization of both dog allergens is still not complete. Their deduced amino acid sequences indicate the presence of three cysteine residues, probably connected with a disulfide bridge. We compared the biochemical and immunological properties of Can f 1 with those of Can f 2 using gel filtration, electrophoresis, and immunological assays. METHODS: The rCan f 1, rCan f 2 and dog salivary proteins containing natural Can f 1 and Can f 2 were analyzed by HPLC gel filtration. The recombinant Can f 1 (rCan f 1) and rCan f 2 were analyzed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with or without reduction. The binding ability of rabbit IgG purified by protein G affinity chromatography from the antiserum against rCan f 1 and rCan f 2 was examined after a reduction in the recombinant allergens. The immunological cross-reaction between rCan f 1 and rCan f 2 was examined by an enzyme-linked immunosorbent assay (ELISA) using the rabbit IgG against rCan f 1 and rCan f 2. The cross-reaction of human IgE in the serum of a patient with dogallergy between rCan f 1 and rCan f 2 was also analyzed by competitive ELISA. RESULTS: The molecular weights of rCan f 1 and of rCan f 2 were 18 and 21 kDa, respectively, using SDS-PAGE under reducing conditions, but the natural Can f 1 and Can f 2 were separated by HPLC gel filtration into fractions containing proteins of 31 and 34 kDa, respectively. rCan f 1 and rCan f 2 migrated as multiple bands (30-100 kDa) in native PAGE in the presence or absence of a reductant. The molecular weights of natural Can f 1 and of Can f 2 were 20 and 23 kDa, respectively, in SDS-PAGE under reducing conditions. The ability of rabbit IgG to bind to rCan f 1 and rCan f 2 increased after the reduction of the recombinant allergens. The rabbit IgG against rCan f 1 bound to rCan f 2. Cross-reaction of human IgE was observed between rCan f 1 and rCan f 2. CONCLUSIONS: In the native and recombinant forms, Can f 1 and Can f 2 possessed a dimer structure under natural (non-reduced) condition. The dimers of Can f 1 and of Can f 2 were not built with a disulfide bridge but by non-covalent association. Cleavage of a disulfide bond of rCan f 1 and rCan f 2 increased the ability of binding of rabbit IgG to the allergens. The cross-reactivity of rabbit IgG and human IgE between rCan f 1 and rCan f 2 indicates that the same epitope(s) was present in Can f 1 and Can f 2.