Affiliations: [a] Department of Rheumatology Research, University College and Middlesex School of Medicine, London, UK | [b] Department of Immunochemistry, CNRS, IBMC, Strasbourg, France
Note: [] Address reprint requests to: Dr. R.A. Watts, Rheumatology Research Unit/Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. A version of this paper was presented at the First International Conference on Human Antibodies and Hybridomas, Orlando, FL, USA, 18–20 April 1990.
Abstract: Human hybridomas were produced by fusion of the GM 4672 cell line with lymphocytes from the peripheral blood, spleen, and bone marrow of patients with systemic lupus erythematosus. Lymphocytes were also obtained from normal human fetal liver of 12 weeks' gestation. The influence of anatomical source, fusion ratio, pre-stimulation with pokeweed mitogen, HLA match, and disease activity at the time of fusion was studied. Supernatants were screened for immunoglobulin secretion and binding to DNA, cardiolipin, poly(ADP-ribose), and histones by enzyme-linked immunoassay. A total of 28 fusions from 14 donors and 6 fusions with fetal lymphocytes were performed. The spleen was found to be the most efficient source of lymphocytes, with a fusion ratio of 1:1 resulting in a maximum yield of 27 clones/107 lymphocytes fused. HLA matching was a factor influencing the outcome with HLA A2 matching being the most important. Pre-stimulation with pokeweed mitogen did not improve the fusion frequency, and fusions using lymphocytes from patients with active disease were only marginally more successful. Over 95% of clones secreted immunoglobulin; autoreactivity was found against DNA, histones, cardiolipin, and poly(ADP-ribose). All hybrids with autoreactivity secreted IgM.
Keywords: human hybridoma, SLE, HLA, autoantibody, monoclonal antibody
