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Ana Carrion, Bijan Etemad-Gilbertson, Jing Zhou, Aditi Soni, Michael Roguska; Characterization of the Stoichiometry of Human Complement C5 Binding to LFG316. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3432.
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© ARVO (1962-2015); The Authors (2016-present)
LFG316 is a fully-human therapeutic IgG that targets complement C5. The goal of this study was to characterize the binding stoichiometry of LFG316 to human C5.
Three methods were used to assess binding stoichiometry; surface plasmon resonance (Biacore), isothermal titration calorimetry (ITC) and ELISA. The Biacore method comprised comparison C5 binding to LFG316 IgG or LFG316 Fab in parallel flow cells under conditions which allowed for sequential binding by the IgG to C5. ITC was performed by titration of LFG316 against C5 and a binding ratio calculated from the exothermic reaction. Finally, a non-competing anti-C5 antibody was used as a capture and detection reagent in a sandwich ELISA format in which binding to C5 was compared for LFG316 IgG and LFG316 Fab antibodies.
In the Biacore analysis, LFG316 that was bound to C5 ligand captured on a biocap chip was able to bind sequentially to C5 analyte, whereas the LFG316 Fab could only bind to the C5 ligand on the chip, indicating 1:2 binding of the IgG. A heat plot generated from the ITC assay yielded a binding ratio of 1:2. In the ELISA, LFG316 IgG bound to two bound C5 molecules could be detected, whereas as expected only a single bound C5 was detected with the LFG316 Fab, also indicating a 1:2 binding ratio.
Using three different analytical methods, Biacore, ELISA, and ITC, we observed a 1:2 binding stoichiometry between LFG316 and human complement C5.
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