IgE antibodies to recombinant forms of Fel d I: Dichotomy between fluid-phase and solid-phase binding studies

Formation of Disulfide Bonds and Homodimers of the Major Cat Allergen Fel d 1 Equivalent to the Natural Allergen by Expression in Escherichia coli

“…The somewhat better homologous inhibition achieved using high concentrations of nFel d 1 is likely to be caused by the inhibition of antibodies present in the serum pool by matching impurities in the nFel d 1 preparation. The mixture of chains 1 and 2 showed significantly lower IgE binding capacity in direct ELISA and a lower specific activity in the inhibition assay, which is consistent with previous findings (32,33) suggesting a distorted protein preparation with fewer exposed epitopes.…”

“…Attempts to reconstitute a mixture of two isolated polypeptides comprising three disulfide bonds produced in E. coli have sometimes been only partially successful (32,33,40). Therefore we engineered a head-to-tail construct of the two polypeptide chains of Fel d 1, which we considered would facilitate a uniform and naturallike folding.…”

“…Cys 3 (1)-Cys 73 (2), Cys 44 (1)-Cys 48 (2), and Cys 70 (1)-Cys 7 (2) (22), suggesting an anti-parallel orientation of Fel d 1 peptides. Several attempts have been made to associate the separate peptides into a native-like allergen in Escherichia coli with only partial success (31)(32)(33), and recently a soluble and immunoreactive chain 1-linker-chain 2 fusion expressed in baculovirus was described (34). A mix of the separate chains has proven to be useful for in vitro allergy diagnosis (12, 33), but to date no soluble, stable, and correctly folded recombinant Fel d 1 (rFel d 1) homodimer with retained disulfide formation has been obtained by expression in E. coli.…”

Distribution of Core Fragments from the Major Cat Allergen Fel d 1 Is Maintained among the Main Anatomical Sites of Production

“…Additionally, chain 2 contains an N-linked oligosaccharide composed of triantennary glycans [11], which has been shown not to be directly involved in antibody recognition of Fel d 1 [12]. Indeed, the main antigenic region is located in the peptidic moiety of the protein [12,13,14] with 4 epitopes identified in recombinant chain 1, using the 6 monoclonal antibodies (mAbs) described so far [15,16] (6F9, 3E4, 1G9, 8F3, 2H4, and 10G7). Interestingly, none of these mAbs was found to bind recombinant chain 2 [16].…”

“…Indeed, the main antigenic region is located in the peptidic moiety of the protein [12,13,14] with 4 epitopes identified in recombinant chain 1, using the 6 monoclonal antibodies (mAbs) described so far [15,16] (6F9, 3E4, 1G9, 8F3, 2H4, and 10G7). Interestingly, none of these mAbs was found to bind recombinant chain 2 [16]. …”