Recombinant Mouse Anti-HCV E2 Antibody (mAb8) (CAT#: PABL-123)

Figure 1 Peptide specificity and binding affinity of the MAbs.

(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.

Figure 2 (A) Neutralization of genotype 1a/2a virus. Each antibody at the concentrations of 0.1, 1, 10, and 100 μg/ml was incubated with the appropriately diluted genotype 1a/2a virus before the mixture was added to Huh 7.5 cells. Cell culture medium (DMEM) was used as a negative control for the antibody. The x axis indicates the particular antibody tested. The y axis indicates the relative infectivity of the virus (%), i.e., the percentage of the negative control.

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.

Figure 3 (C) Inability of the antibodies to cross-neutralize the J6/JFH1 virus. The antibodies were tested for their abilities to neutralize J6/JFH1, a genotype 2a virus, in Huh 7.5 cells with the procedure described in panel A.

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.

Figure 4 Epitope mapping by screening random peptide phage display libraries.

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.

Figure 5 Use of mutational analysis to identify the residues that are critical for antibody recognition.

(A) Biotin-conjugated peptides were chemically synthesized to represent the truncated peptide B, i.e., B short, from the E2 protein of HCV genotype 1a (H77 strain) and its mutations. The B short mutant peptides contained a single alanine substitution at positions 437, 438, 440, 441, and 442, respectively. A hyphen indicates an amino acid residue identical to that of the H77 sequence. (B) Biotin-conjugated B short peptide and its mutants were added to streptavidin-coated 96-well plates at 200 ng/well in an ELISA. Each MAb (ascites fluid) was diluted 1:105 dilution, and applied as the primary antibody. The x axis indicates the antibodies used in the assay. The y axis indicates the absorbance at 405 nm, representing specific binding of a given antibody to each individual peptide.

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.