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

互联网2014-02-13

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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has allowed us to produce human hybridomas secreting 1–200 �g/mL of IgG specific for HCMV (3 ), HTLV-I (4 ), and HCV (unpublished) using several different mouse-human heteromyeloma fusion partners.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.