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. Author manuscript; available in PMC: 2018 Jul 25.
Published in final edited form as: ACS Nano. 2017 Jul 12;11(7):7110–7117. doi: 10.1021/acsnano.7b02755

Figure 2.

Figure 2

H5 antibody recognizes conformational change of the integrin-binding domain by binding an epitope specifically on the ninth type III repeat that can be exposed by denaturation. (A,B) SPR analysis of binding of H5 antibody to recombinant FnIII9*10 (A) and FnIII9-4G-10 (B) fragments demonstrates the conformation selectivity of its binding. H5 binds preferentially to the molecularly extended conformation. Black lines show experimental sensogram traces, and red lines show fitted data. Equilibrium dissociation constants (KD) are shown above the curves. (C) Competitive binding of the H5 antibody to FnIII9-4G-10 in the presence of soluble FnIII9 (FnIII6-9) and FnIII10 (FnIII10-14) fragments. FnIII9-4G-10 was immobilized on ELISA plates, and the H5 antibody was co-incubated with the indicated soluble Fn fragments prior to incubation with FnIII9-4G-10. N = 8 for each group; error bars reflect SEM. (D,E) Domain mapping studies were performed by SPR using Fn fragments including either the ninth type III repeat (FnIII6-9) (B) or the 10th type III repeat (FnIII10-14) (C). Binding of H5 was only observed by SPR when fragments including the ninth Fn type III repeat were immobilized, suggesting that the epitope for H5 is located within FnIII9. (F) ELISA of H5 binding to full-length Fn and Fn fragments. H5 bound increasingly to increasing amounts of surface-adsorbed FnIII9-4G-10 to a significantly greater degree than FnIII9*10 or full-length Fn at all concentrations (p < 0.0001) (two-way ANOVA with Tukey’s post-test, N = 3). (G,H) Nitrocellulose dot blot (G) and Western blot (H) of H5 binding to full-length Fn and Fn fragments. Under these denaturing conditions, H5 binds FnIII9-4G-10 and FnIII9*10 to a similar degree. (I–K) ELISA (I), dot blot (J), and Western blot (K) of HFN7.1, a commercially available antibody targeting FnIII9-10. In the ELISA, HFN7.1 only bound FnIII9-4G-10 to a significantly greater degree than FnIII9*10 at 1 μM (p < 0.05) and was not significant at higher concentrations (two-way ANOVA with Tukey’s post-test, N = 3). HFN7.1 did not show preferential affinity for FnIII9*10 or FnIII9-4G-10 in the dot blot or Western blot.