摘要
近年来,细胞电融合技术(Electrofusion)得到了迅速发展和应用.特别是在单克隆抗体领域,人们希望能以电融合技术的高融合频率来更有效地获得分泌单抗的杂交瘤.现主要有两种较有效的电融合方法来实现这一目的:一是Nova/Hopkins的抗原—抗生物素—生物素桥联(antigen-avidin-biotin bridge)方法,另一种则是Zimmermann电融合法.Nova/Hopkins方法虽然非常有效。
Electrofasion between mouse (BALB/C) Lymphocytes and SP2/0myeloma cells was performed in tn isotonic Hepes-buffered Saline solution (HBS:10 mM Hepes, 140 mM NaCl, 8 mM KC1, 0.5 mM Mg-acetate, 0.1 mM Ca-ac-etate, 0.1 mM MnCl2, pH = 7.2), in which the cells were agglutinated by Con-canavalin A (25μg/ml), After being discharged by a 0.5μF capacitor three times (pulse amplitude = 4KV/cm, pulse time constant=6μs, pulse interval= 1 sec),the cells were incubated at 37℃ for 10 min and then transfered into a modified balanced salt solution containing α-D-glucose and α-D-mannose (PBS-GM: 9.6mM Na2HPO4, 1.5mM KH2PO4, 8mM KC1, 32mM NaCl, 0.1M Glucose, 0.1M Mannose, 0.5mM Mg-acetate, 0.1mM Ca-acetate, 0. 1mM MnCl2 ,pH= 7.2) for 30 min incubation at 37℃, to round-off the fusion products and get rid of ConA, High hybridoma yield, about 76±32 colonies per well/2.25 × 105 myeloma cells, could be obtained 7 days after fusion, and half of the colonies were composed of more than ten cells, some of them (about 10%) were composed of hundreds of cells.
出处
《生物物理学报》
CAS
CSCD
北大核心
1989年第1期94-95,共2页
Acta Biophysica Sinica
