title: Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras.
abstract: Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes.
authors.affiliation: 1 Swedish Orphan Biovitrum AB , Stockholm, Sweden .
authors.forename: Bartek
authors.initials: B
authors.lastname: Zuber
authors.affiliation: 2 Mabtech , Nacka Strand, Sweden .
authors.forename: Karin
authors.initials: K
authors.lastname: Rudström
authors.affiliation: 2 Mabtech , Nacka Strand, Sweden .
authors.forename: Cecilia
authors.initials: C
authors.lastname: Ehrnfelt
authors.affiliation: 2 Mabtech , Nacka Strand, Sweden .|3 Department of Immunology, Stockholm University , Stockholm, Sweden .
authors.forename: Niklas
authors.initials: N
authors.lastname: Ahlborg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002417:Cattle; D042783:Endothelial Cells; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D051379:Mice; D009500:Neutralization Tests; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D017471:Receptors, Interferon / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D000096743:Interferon gamma Receptor
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000939:Epitopes; D007371:Interferon-gamma; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D000096743:Interferon gamma Receptor