Mutation of acidic residues of Lu gp reduces Ln511/521 binding. (A) Representative ELISA titrations of 5, 0.5, 0.05, and 0.005 nM Lu gp binding to 5 nM Ln511/521 for the E309A, D310A, D312A, and D315A (solid line) mutations in comparison with native Lu gp (dashed line). Standard deviation for each point is less than 0.1. ELISA results of all other mutations can be seen in Figure S3. (B) The level of binding to Ln511/521 of mutant Lu gp compared with the native protein at 0.05 nM measured in an ELISA. Proteins were assayed in duplicate and the results shown are the mean of 2 separate ELISA plates and are expressed as the percentage of the absorption seen from wells containing native Lu gpFc (OD₄₅₀ = 0.95). (C) Ln511/521 binding assessed by surface plasmon resonance using a Biacore X. Shown is the mean (± SEM) change in response units (RU) over the course of a 100-μL injection of 10 nM Ln511/521 for 2 assays per protein. In both panels B-C, Ln511/521 binding to Muc18 acted as a negative control. (D) Overlaid sensorgrams for a representative sample of the different proteins showing the association and dissociation curves of Ln511/521 binding to Lu gpFc. Vertical arrows indicate the beginning and end points of the Ln511/521 injection.