a. a. 35–47 mediate the binding of mCRP to CFH in ELISAs a.a. 35–47 mediate the binding of mCRP to CFH in ELISAs. (A) Urea-denatured mCRP, recombinant Cys-mutated mCRP, or native CRP at the indicated concentrations was added to CFH immobilized onto microtiter wells. a.a. 35–47 mediate the binding of mCRP to CFH in ELISAs. (A) Urea-denatured mCRP, recombinant Cys-mutated mCRP, or native CRP at the indicated concentrations was added to CFH immobilized onto microtiter wells. Cys-mutated mCRP represents an mCRP conformation with enhanced activities.19 The binding was detected with mCRP-specific mAb 3H12 or native CRP-specific mAb 1D6.18,20 mCRP bound strongly to CFH, whereas native CRP did not. (B) Urea-denatured mCRP or (C) Cys-mutated mCRP was added to immobilized CFH together with the indicated CRP peptides (n=3). Prominent inhibition of mCRP binding was only observed with a.a. 35–47. (D) a.a. 35–47 with a C-terminal biotin tag were added to immobilized CFH (n=3). Peptide binding was determined with HRP-labeled streptavidin. This confirmed the direct binding of a.a. 35–47 to CFH. (E) Wild-type and mutant mCRP lacking a.a. 35–47 were expressed in Escherichia coli. These proteins were purified, and their binding to immobilized CFH was examined (n=3). Wild-type mCRP showed strong binding as expected, whereas the binding capacity was lost on deletion of a.a. 35–47. (F) Purified anti-a.a. 35–47 but not anti-a.a. 199–206 autoantibodies inhibited the binding of mCRP to CFH. *P<0.05. Qiu-yu Li et al. JASN 2017;28:3044-3054 ©2017 by American Society of Nephrology