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. 2014 Dec 23;290(7):4022–4037. doi: 10.1074/jbc.M114.617787

FIGURE 2.

FIGURE 2.

A–D, binding of hTNFR1 and inhibition of TNF/hTNFR1 signaling by the monovalent nanobodies and surface plasmon resonance sensorgrams of Nb 96 and Nb 70 binding to immobilized hTNFR1. A, to determine the binding affinity, an hTNFR1 ELISA with the monovalent Nbs was performed. A serial 0.2 dilution was applied, starting at 2.7 μm. B, using a HEK-2 blue assay, the inhibition capacity of TNF/hTNFR1 signaling by the monovalent Nbs was determined. HEK-2 blue cells were preincubated with a 0.2 Nb dilution series starting at 2.7 μm and stimulated with 1000 IU/ml TNF. Nb 70 was identified as the only inhibiting Nb. C and D, SPR analysis of Nb 96 and Nb 70. The adjusted sensorgrams overlays show binding of Nb 96 or Nb 70 applied in a dilution series from 1.95 to 500 nm to immobilized hTNFR1. Dotted lines show global fitting of the binding data to a 1:1 interaction model. Both Nbs have good association constants (high ka), but show quick dissociation (kd). Nb Alb-Ctrl-Ctrl, an irrelevant control Nanobody; hTNFR1 Ab, a human TNFR1 antibody, positive control. Black dotted line, Nanobody concentration that binds 50% of hTNFR1 or albumin. The ELISA and HEK-2 blue assay were done in triplicate, and data are represented as mean ± S.E. Surface plasmon resonance analyses were done in duplicate.