Cao Y, Vinayagamoorthy T, Noujaim A A, Suresh M R
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
J Immunol Methods. 1995 Nov 16;187(1):1-7. doi: 10.1016/0022-1759(95)00160-c.
A simple non-selective methodology was developed and standardized to generate desired hybrid-hybridoma or quadroma secreting bifunctional antibodies. This novel protocol is based on microelectrofusion on a meander chamber using a few hundred cells of each of the two parental hybridomas with no laborious drug selection procedures. Seeding approximately 10 cells per well in a 96-well microtitre plate after fusion in 200 microliters standard medium containing 20% FBS and 10% Origen growth factor generated positive quadromas secreting bispecific antibodies with good stability after the second reclone. Compared to the conventional PEG fusion and other methods this simple protocol is both rapid and economical. Generally, conventional methods to make quadromas and triomas require the introduction of drug selection markers into one or both of the parental cells, a procedure that could take 3-6 months. Utilizing the non-selective microelectrofusion method described here, we have generated several quadromas in a very short time. Further, such a protocol could also be potentially adopted to generate human hybridomas with few B cells isolated from peripheral blood lymphocytes enriched by antigen specific panning or affinity microelectrofusions.
我们开发并标准化了一种简单的非选择性方法，以产生所需的分泌双功能抗体的杂交 - 杂交瘤或四瘤。这种新方案基于在曲折腔室中进行的微电融合，使用两种亲本杂交瘤中的每一种的几百个细胞，无需繁琐的药物筛选程序。在含有20%胎牛血清和10%Origen生长因子的200微升标准培养基中融合后，在96孔微量滴定板中每孔接种约10个细胞，经过第二次亚克隆后产生了分泌双特异性抗体且稳定性良好的阳性四瘤。与传统的聚乙二醇融合法和其他方法相比，这个简单的方案既快速又经济。一般来说，制备四瘤和三瘤的传统方法需要将药物选择标记引入一个或两个亲本细胞中，这一过程可能需要3至6个月。利用本文所述的非选择性微电融合方法，我们在很短的时间内产生了几个四瘤。此外，这样的方案也有可能被用于从通过抗原特异性淘选或亲和微电融合富集的外周血淋巴细胞中分离出少量B细胞来产生人杂交瘤。
