Generation of a human monoclonal antibody to hepatitis C virus, JRA1 by activation of peripheral blood lymphocytes and hypo-osmolar electrofusion.

Abstract

We have generated a human monoclonal antibody with binding specificity for hepatitis C virus (HCV)-specific peptides using peripheral blood lymphocytes isolated from a HCV antibody positive patient. The B-lymphocytes were stimulated with lipopolysaccharide (LPS) for 72 hours prior to the fusion. A recently described high efficiency hypo-osmolar electrofusion technique was employed, allowing generation of a large number of human hybridomas. The hybridomas were screened for human immunoglobulin and HCV-specific peptide binding by EIA. A single HCV-positive clone, JRA1, was detected and sub-cloned. Isotype analysis showed it to secrete an IgM lambda monoclonal antibody. The antibody was positive on both first and second generation HCV antibody analysis. This study confirms that viable pathogen-specific B-cells may be recovered from the peripheral blood. Although such cells are likely to be relatively uncommon in the circulating B-cell pool, they may be successfully immortalized by high efficiency electrofusion techniques. This technique might be valuable for the generation of human monoclonal antibodies with specificity for other human pathogens.