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 isolatedfrom 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 electro fusion technique was employed, allowing generation of a large number of human hybridomas. The hybridomas were screenedfor 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 λ 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 recoveredfrom 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 electro fusion techniques. This technique might be valuable for the generation of human monoclonal antibodies with specificity for other human pathogens.