BACKGROUND: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule. OBJECTIVE: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells. METHODS: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22(+)) or alone (rFel d 1 H22 (-)). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22 (+) to the CD64 receptor on leukocyte subpopulations and on the THP -1 cell line was analyzed by means of flow cytometry. RESULTS: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22(+)) and 22 kd (rFel d 1 H22 (-)). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = > 0.72,P <.001). The rFel d 1 H22(+) bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22(+) to THP-1 cells was inhibited by a soluble CD64 fusion protein. CONCLUSIONS: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunoreactivities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.
BACKGROUND: Cat allergen Fel d 1 is a heterodimer encoded by 2 separate genes that has been difficult to produce as a fully immunoreactive molecule. OBJECTIVE: We sought to engineer recombinant (r) Fel d 1 with IgE and IgG antibody binding comparable with that of the natural allergen that could be targeted to antigen-presenting cells. METHODS: The rFel d 1 chains were coexpressed in baculovirus, either linked to the anti-CD64 antibody H22 (rFel d 1 H22(+)) or alone (rFel d 1 H22 (-)). Binding of expressed allergens to mouse and human antibodies was compared with that of natural (n) Fel d 1 by means of enzyme immunoassay and antigen-binding and inhibition RIAs. Binding of rFel d 1 H22 (+) to the CD64 receptor on leukocyte subpopulations and on the THP -1 cell line was analyzed by means of flow cytometry. RESULTS: The baculovirus-expressed allergens migrated with molecular weights of 49 kd (rFel d 1 H22(+)) and 22 kd (rFel d 1 H22 (-)). The rFel d 1 inhibited IgG antibody binding to nFel d 1 by greater than 95% and showed identical dose-dependent inhibition curves. There was an excellent quantitative correlation between IgE and IgG antibody binding to rFel d 1 and nFel d 1 in sera from patients with cat allergy (IgE: n = 258, r = > 0.72,P <.001). The rFel d 1 H22(+) bound to monocytes but not to lymphocytes or neutrophils, and binding of rFel d 1 H22(+) to THP-1 cells was inhibited by a soluble CD64 fusion protein. CONCLUSIONS: Recombinant Fel d 1 chains have been successfully coexpressed as mature proteins with comparable immunoreactivities to nFel d 1. The rFel d 1 can be targeted to antigen-presenting cells through CD64. These constructs will facilitate structural studies of Fel d 1 and the development of improved allergy diagnostics and therapeutics.
Authors: Serge A Versteeg; Ingrid Bulder; Martin Himly; Toni M van Capel; R van den Hout; Stef J Koppelman; Esther C de Jong; Fatima Ferreira; Ronald van Ree Journal: World Allergy Organ J Date: 2011-07-14 Impact factor: 4.084