Generation of a human monoclonal antibody to hepatitis C virus, JRA1 by activation of peripheral blood lymphocytes and hypo-osmolar electrofusion.
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.
D000328:Adult; D000914:Antibodies, Viral / Q000276:immunology*; D002459:Cell Fusion; D016174:Hepacivirus / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000166:cytology*; D008213:Lymphocyte Activation; D008297:Male
D000914:Antibodies, Viral