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        Stabilizing Antibody Secretion of Human Epstein Barr Virus-Activated B-Lymphocytes with Hybridoma Formation by Electrofusion

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        1808
        Epstein Barr virus (EBV) can be used to transform human B-lymphocytes to derive populations of cells secreting specific antibodies of interest. Isolating monoclonal or stable populations of these cells, however, has proven very difficult (1 ). In our laboratory, we have developed methods to immortalize specific antibody-producing cells by fusing secreting EBV-activated lymphocytes to mouse-human heteromyeloma cell lines with electrofusion, followed by cloning (2 ). This methodology has allowed us to produce human hybridomas secreting 1–200 �g/mL of IgG specific for HCMV (3 ), HTLV-I (4 ), and HCV (unpublished) using several different mouse-human heteromyeloma fusion partners. Because as few as 5 � 104 –106 EBV-activated B-cells can be successfully fused with a high degree of efficiency and consistency (up to one hybrid for each 100–1000 input EBV-activated cells), they can be fused as soon as antibody can be detected in a microtiter well, before the cells lose secretion or are overgrown by nonsecreters (5 ). High efficiency is achieved by varying the electrical parameters depending on the specific cells, the cell number, and the medium in which the cells are fused (see Table 1 ).
        Table 1  Examples of Fusion Voltage Used with Different Fusion Partners in Different Fusion Media to Immortalize Antigen-Specific Antibody Secreted by EBV-Activated Lymphocytes from Peripheral Blood

        Hybrid name

        Antibody secreted to

        IgG, �g/mL

        # EBV fused

        Heteromyeloma fusion partner

        Fusion medium

        dc Fusion voltage

        Range in hybridoma formation efficiency (5 )

        Z10 (18 )

        CMV

        5

        2 � 106

        SBC-H2O

        Iso-osmolar (14 )

        3.0 kV/cm 3 pulses/15 �s

        34–68

        X2-16 (19 )

        CMV

        100

        1.4 � 106

        K6 H6 /B5

        Iso-osmolar (14 )

        3.0 kV/cm 3 pulses/15 �s

        6–18

        IH-9 (20 )

        HTLV-I

        3

        105

        K6 H6 /B5

        Hypo-osmolar 75L3

        1.0 kV/cm 3 pulses/15 �s

        141–258

        WA-04 2B10 (4 )

        HTLV-I

        40

        1.5 � 105

        H73C11

        Hypo-osmolar 100L3

        1.25 kV/cm 1 pulse/10 �s

        57–283

        WA-11 1F5 (4 )

        HTLV-I

        70

        1.5 � 106

        K6 H6 /B5

        Iso-osmolar 300L3

        1.75 kV/cm 3 pulses/15 �s

        40–96

        JB-04 1D7

        HCV

        90

        106

        K6 H6 /B5

        Iso-osmolar 300L3

        1.75 kV/cm 3 pulses/15 �s

        11–66

        JB-16 2D6

        HCV

        14

        1.5 � 105

        H73C11

        Hypo-osmolar 100L3

        1.25 kV/cm 1 pulse/10 �s

        194–388

        JB-17 1D2

        HCV

        60

        1.5 � 105

        K6 H6 /B5

        Hypo-osmolar 100L3

        1.25 kV/cm 3 pulses/15 �s

        80–200

        a (The number of wells with growth � The range in colony#/well in over 1/2 the wells) / 105 input EBV-activated B-cells.
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