Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-gamma Using Human-Bovine Interferon-gamma Chimeras.

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Zuber B, Rudstrom K, Ehrnfelt C, Ahlborg N

Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-gamma Using Human-Bovine Interferon-gamma Chimeras.

J Interferon Cytokine Res. 2016 Sep;36(9):542-51. doi: 10.1089/jir.2016.0017. Epub 2016 Jun 23.

PubMed ID
27336613 [ View in PubMed
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Abstract

Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-gamma. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-gamma 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-gamma, epitopes were identified using 7 h/bIFN-gamma chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-gamma 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-gamma-mediated activation of human cells, in line with the involvement of region A in the IFN-gamma 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.

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