Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate (original) (raw)
Figure 3
HC-84 HMAbs bind to conformational epitopes that are not within antigenic domains A and B.
(A) Antibody binding to H77C (wt) and D535A recombinant E2 lysates by ELISA. The assays were performed with 1 µg of E2/ml that was captured by GNA pre-coated wells, and followed by incubation with each HMAb at 1 µg/ml (_x_-axis). Positive control is HC-1, an antigenic domain B HMAb [27] and negative control is R04. The _y_-axis shows the mean optical density (O.D.) values. Data are derived from triplicate wells, the mean of two experiments ±SD. (B) Immunoprecipitation of 1a H77C recombinant E1E2 lysate by each HCV-84 HMAb (as indicated at the top of the panel). HC-1, an antigenic domain B HMAb, was used as a positive control and R04 was used as a negative control. The immunoprecipitated pellet was separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis under reducing conditions, and immunoblots were analyzed with HMAbs recognizing linear epitopes: anti-E2, HC-33.1 [45] and anti-E1, H-111 [49]. (C) HC-84 HMAbs do not bind to denatured 1a HCV E2. Recombinant E1E2 lysate was either left untreated (black bars) or denatured by incubation with 0.5% sodium dodecyl sulfate and 5 mM dithiothreitol for 15 min at 56°C (red bars). After treatment, the proteins were diluted 1∶5 in BLOTTO and captured by pre-coated GNA wells. After washing and blocking, bound proteins were incubated with each HC-84 HMAb at 5 µg/ml (_x_-axis) and a control HMAb, HC-33.1 [45]. Bound antibody was detected as described in Materials and Methods. The _y_-axis shows the mean optical density values for triplicate wells, the mean of two experiments ±SD.