Human Anti-HCV NS3 Recombinant Antibody (clone Hfab-aNS3) (CAT#: FAMAB-1778CQ)

Figure 1 Expression of human Fab antibody (HFab-aNS3) in Huh-7 cells.

Huh-7 cells were transfected with pFab-CMV-NS3 (H+L) plasmid using FuGENE 6 reagent and after 72 hours the cells were examined for the expression of Fab by immunocytochemistry. Transfected Huh-7 cells were immobilized on glass slides by cytospin, reacted with biotin-labeled anti-human F(ab)2 antibodies, and reacted with ABC reagents. Color was developed with DAB. Cytoplasmic expression of antibody in the transfected cells (brown staining, shown by arrows) was present in the majority of cells transfected with Fab expression plasmid. Mock-transfected Huh-7 cells were negative.

Prabhu, R., Khalap, N., Burioni, R., Clementi, M., Garry, R. F., & Dash, S. (2004). Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone. The American journal of pathology, 165(4), 1163-1173.

Figure 2 Intracellular expression of Fab inhibits full-length HCV replication.

HepG2 cells were transfected with 10 μg of in vitro transcribed full-length HCV RNA using the DEAE-dextran method. A week later, cells were transfected with antibody expression plasmid (HFab-aNS3). RNA extracts were prepared from transfected cells at 3, 6, 9, and 12 days and HCV RNA levels were measured using competitive reverse transcriptase-polymerase chain reaction assay. Intracellular HCV RNA levels were reduced significantly after antibody transfection without any alteration in the GAPDH mRNA level.

Prabhu, R., Khalap, N., Burioni, R., Clementi, M., Garry, R. F., & Dash, S. (2004). Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone. The American journal of pathology, 165(4), 1163-1173.

Figure 3 Affinity-purified human Fab antibody inhibits helicase of NS3 protein in a concentration-dependent manner.

A: Helicase assay was performed in vitro in the presence of different concentrations of unrelated Fab antibody control. Control Fab did not inhibit helicase activity of NS3 protein. B: Helicase assay was performed in vitro in the presence of different concentrations of human Fab to NS3 (HFab-aNS3) antibody. Helicase activity can be inhibited by human Fab at 2:1 molar ratio.

Prabhu, R., Khalap, N., Burioni, R., Clementi, M., Garry, R. F., & Dash, S. (2004). Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone. The American journal of pathology, 165(4), 1163-1173.

Figure 4 Intracellular expression of HFab-aNS3 inhibits HCV-negative strand.

Huh-7 cells were infected with a replication-defective adenovirus carrying T7 RNA polymerase gene. These cells were then co-transfected with transcription plasmid and antibody expression plasmid. After 72 hours, RNA extracts were prepared, digested with DNase, and tested for negative strand HCV RNA by RPA assay. Top: RPA for HCV-negative strand RNA in the cells transfected with HFab-aNS3. Bottom: RPA for GAPDH mRNA. Lane 1, Probe alone; lane 2, probe digested with RNase; lane 3, cells transfected with pMO9.6T7-Rz alone; lane 4, cells co-transfected with pMO9.6T7-Rz and pFab CMV (control); lane 5, cells co-transfected with pMO9.6T7-Rz and pFab-CMV-NS3 (H+L).

Prabhu, R., Khalap, N., Burioni, R., Clementi, M., Garry, R. F., & Dash, S. (2004). Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone. The American journal of pathology, 165(4), 1163-1173.