Recombinant Mouse Anti-HCV E2 Antibody (mAb12)

(A) Peptides used for the present study. Peptide A corresponding to amino acid residues 412 to 447 of the E2 protein of HCV H strain (H77) was used to immunize mice for generating the MAbs tested in the present study. The truncated forms of peptide A, i.e., peptide B, B short, and peptide D, are indicated. The locations of epitope I and epitope II within peptide A, as mapped in our previous studies, are shown. (B) Peptide A specificity of the MAbs in an ELISA. Biotin-conjugated peptide A was added to streptavidin-coated 96-well plates (200 ng/well). Each MAb (ascites fluid) was diluted 1:1,000 and used as the primary antibody. The y axis indicates the absorbance at 405 nm obtained in the ELISA, representing specific binding of a given antibody to peptide A. The data shown represent three independent experiments. (C) Peptide B specificity of the MAbs in an ELISA. Instead of peptide A, biotin-conjugated peptide B was used for the ELISA as described in panel B. (D) The affinities of B-specific antibodies to peptide A and B were determined by an ELISA using sodium thiocyanate (NaSCN) as a chaotropic agent as described in reference 22. An ELISA was performed with 1:20,000-diluted ascites fluid in the presence or absence of various concentrations of NaSCN as indicated. The specific binding affinity was calculated based on the values obtained with or without NaSCN.

Duan, H., Kachko, A., Zhong, L., Struble, E., Pandey, S., Yan, H.,... & Major, M. (2012). Amino Acid Residue-Specific Neutralization and Non-neutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein. Journal of virology, JVI-00994.

Duan, H., Kachko, A., Zhong, L., Struble, E., Pandey, S., Yan, H.,... & Major, M. (2012). Amino Acid Residue-Specific Neutralization and Non-neutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein. Journal of virology, JVI-00994.

Duan, H., Kachko, A., Zhong, L., Struble, E., Pandey, S., Yan, H.,... & Major, M. (2012). Amino Acid Residue-Specific Neutralization and Non-neutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein. Journal of virology, JVI-00994.

Duan, H., Kachko, A., Zhong, L., Struble, E., Pandey, S., Yan, H.,... & Major, M. (2012). Amino Acid Residue-Specific Neutralization and Non-neutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein. Journal of virology, JVI-00994.

HCIGIV at the ID50 (1:3,000 dilution) was used to neutralize genotype 1a/2a virus in the presence or absence of different concentrations of antibody 12 (A) or antibody 50 (B). The cell culture medium (Med) was used as the negative control. (C) The ability of antibody 41 to neutralize genotype 1a/2a virus was measured in the presence or absence of nonneutralizing antibody 12 or antibody 50 according to the procedure described in Fig. 3. Antibody 41 was diluted at 1:400 in DMEM, and then mixed with antibody 12 or antibody 50 with the ratios of 1:1 and 1:4 (vol/vol). The antibody mixture was subsequently incubated with genotype 1a/2a chimeric virus at 37°C for 1 h before being added to Huh 7.5 cells. The x axis indicates the samples used in this assay. The y axis indicates the relative infectivity of the virus (%).

Duan, H., Kachko, A., Zhong, L., Struble, E., Pandey, S., Yan, H.,... & Major, M. (2012). Amino Acid Residue-Specific Neutralization and Non-neutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein. Journal of virology, JVI-00994.

The amino acid sequences of phage clusters identified after at least three rounds of screening phage-display libraries (12-mer and 7-mer) with neutralizing antibodies 8 and 41 and nonneutralizing antibodies 12 and 50 are listed. The numbers of peptides identified over the total numbers of peptides sequenced are shown in parenthesis. The candidate core residues at the epitope-paratope contact interfaces are indicated in bold font. The letter "X" denotes any amino acid residue other than L at that position.

Duan, H., Kachko, A., Zhong, L., Struble, E., Pandey, S., Yan, H.,... & Major, M. (2012). Amino Acid Residue-Specific Neutralization and Non-neutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein. Journal of virology, JVI-00994.