Epitope analysis of the allergen ovalbumin (Gal d II) with monoclonal antibodies and patients' IgE.
Ovalbumin (OVA) is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food. Further knowledge of the antigenic and allergenic epitopes of allergens will provide better treatment of this disease. To analyse these epitopes we produced a panel of monoclonal antibodies (mAbs) against native OVA. The initial information about the epitopes was obtained with the binding patterns of these mAbs in IEF-immunoprints and western blots of OVA under reducing and non-reducing conditions. It was possible to demonstrate that the different conformations of OVA exhibit different epitopes, and that there are other epitopes which are shared by each conformation. Seven different, although sometimes overlapping epitopes, could be determined on native OVA; four different epitopes on denaturated non-reduced OVA by means of immunoblots of the intact molecule. The number of epitopes which could be differentiated by the mAbs was increased by the use of peptide blots after CNBr fragmentation of the molecule. IgE binding to different OVA conformations and to CNBr-fragments of OVA was also detectable and appears in the same regions as the reactivity of some mAbs. Western blots of OVA and CNBr-peptides demonstrate that some antigenic/allergenic binding sites seem at least partly to be continuous epitopes. The identification of the CNBr-fragments was performed by a microsequence analysis of blotted CNBr-fragments after a 2-dimensional electrophoresis. IgE was found to bind the two largest CNBr-fragments (residues 41-172 and 301-385), but not the fragment corresponding to residues 173-196. A number of monoclonal antibodies also reacted with the two large fragments, especially with fragment 301-385, and some bind also to shorter peptides, such as fragment 173-196, which were not reactive to patients' IgE. Most of the monoclonal antibodies and patients' IgE bind to the fragments 41-172 and 301-385 in 2D-PAGE blots suggesting that these fragments are involved in an immunogenic structure.
Kahlert
H
H
Division of Allergology, Forschungsinstitut Borstel, Germany.
Petersen
A
A
Becker
W M
WM
Schlaak
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007525:Isoelectric Focusing; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007073:Immunoglobulin E; D010047:Ovalbumin