Antibody producing human-human hybridomas. I. Technical aspects |
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Authors: | L. Olsson H. Kronstrøm A. Cambon-De Mouzon C. Honzik T. Brodin B. Jakobsen |
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Affiliation: | 1. Medical Department A, State University Hospital, Copenhagen, Denmark;2. Cancer Biology Research Laboratory, Stanford University School of Medicine, Stanford, CA 94305, U.S.A.;3. The Wallenberg Laboratory, University of Lund, Lund, Sweden;4. Tissue-Typing Laboratory, State University Hospital, Copenhagen, Denmark |
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Abstract: | Technical aspects of generation of antibody-secreting human-human hybridomas are evaluated as based on 100 human-human fusions with a human B-lymphoma cell line (RH-L4) or the SKO-007 myeloma cell line as malignant fusion partners, and compared with similar fusion conditions in the mouse hybridoma system. The yield of hybrids was significantly lower when normal peripheral blood lymphocytes were used as fusion partners as compared with spleen lymphocytes, but could be substantially improved by increasing the amount of mitotic active B-lymphocytes by mitogen stimulation of the lymphocytes, preferably in HAT medium, prior to fusion. Furthermore, human hybrids grew slower and had a higher degree of chromosomal instability than usually observed in the mouse hybridoma system. Thus, out of 72 fusions, only 3 stable hybrids with antibody production against a predefined antigen were established. The importance of improved sources of human B-lymphocytes for human-human hybridoma production is discussed and methods of obtaining such improvement suggested. |
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Keywords: | monoclonal antibody human-human hybridoma EBV Epstein-Barr virus ELISA enzyme-linked immunosorbent assay FACS fluorescence-activated cell sorter FCS fetal calf serum FITC fluorescein isothiocyanate HAT medium RPMI 1640 medium with hypoxanthine aminopterin thymidine LPS lipopolysaccharide mPBLs murine peripheral blood lymphocytes NP-40 Nonidet P-40 PBLs peripheral blood lymphocytes PBS phosphate-buffered saline PEG polyethylene glycol PWM pokeweed mitogen SDS sodium dodecyl sulfate SRBC sheep red blood cells |
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