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.
Highlights
Human interferon-g is predominantly produced by T cells and natural killer (NK) cells, activated by immune and inflammatory stimuli, and promotes both protective innate and adaptive immune responses
Utilizing the monoclonal antibodies (mAbs)’ lack of reactivity with bovine (b) IFN-g, epitopes were identified using 7 h/bIFN-g chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-g residues
When analyzed by sandwich ELISA, the mAbs clustered into 2 major groups where antibodies in each group could bind to IFN-g simultaneously and without interference from each other, indicating recognition of distinct epitope regions (Fig. 2)
Summary
Human interferon (hIFN)-g is predominantly produced by T cells and natural killer (NK) cells, activated by immune and inflammatory stimuli, and promotes both protective innate and adaptive immune responses. It is, involved in various immunopathological conditions and aberrant levels of IFN-g are associated with a number of autoinflammatory and autoimmune diseases ( Jager and others 2010; Reinhardt and others 2015). The dimeric nature of the protein has been confirmed by X-ray crystallography showing that IFN-g is primarily helical, with each monomer consisting of 6 alpha-helices (A-F) connected by short loops (Fig. 1A; Ealick and others 1991; Walter and others 1995). The dimer is formed when the C-terminal helices (E and F) from one chain associate head-to-tail with the N-terminal helices A, B, C, and D from the other chain
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