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Binding properties of HC33 HMAbs. (A) Antibody binding to native or denatured recombinant E2 by ELISA. Recombinant E1E2 cell lysates were either left untreated (native) or denatured by boiling for 5 min in TBS and 10% FCS containing 1.0% sodium dodecyl sulfate and 50 mM dithiothreitol (denatured). After treatment, the proteins were diluted 1:5 in BLOTTO and captured by precoated GNA wells. Bound proteins were incubated with each HC33 HMAb at 5 μg/ml (x axis), and bound antibody was detected by antihuman antibody, as described in Materials and Methods. The y axis shows the mean optical density (O.D.) values for triplicate wells, the means of results from three experiments plus or minus SD. (B) Measurement of HC33 scFv affinity to HCV 1a E2 by SPR (as represented by HC33.29). Association and dissociation curves were obtained for each HC33 scFv against immobilized 1a H77C E2 captured by CBH-4D as measured by BIAcore 3000. Each scFv was evaluated at concentrations ranging from 1,000 nM to 31.35 nM (with 2-fold serial dilutions). Data were analyzed as described in Materials and Methods. (C) Summary of the Kon, Koff, and KD values for each HC33 HMAb, HC84.20, and HC84.26. (D) Epitope mapping of the neutralizing HC33 HMAbs. E2 mutant proteins were expressed in HEK 293T cells, and cell lysates were analyzed by ELISA. HC33.1, HC33.8, and HC33.29 were tested at 0.1 μg/ml, HC33.4 at 0.05 μg/ml, and HC33.32 at 0.6 μg/ml. Individual protein expression was normalized by binding of CBH-17, an HCV E2 HMAb, to a linear epitope (44). Two regions of E2 protein were analyzed: aa 412 to 423 and aa 434 to 446. Red indicates 0 to 20%, orange 21 to 40%, brown 41 to 60%, white 61 to 100%, and green >100% binding when the residue was replaced by alanine (or glycine at aa 439), relative to binding to wt. Structurally intact conformation of mutated E2 was defined by control antibodies, CBH-4D and CBH-7, to two different conformational antigenic domains on E2 (35, 44). Retention of binding by these antibodies is necessary to ensure native E2 structure. Data are shown as the mean values of two experiments performed in triplicate.