半固体培养基法制备单抗的可行性研究

目的建立稳定的甲基纤维素半固体培养基一步法筛选和克隆化杂交瘤细胞的方法。方法常规细胞融合后，用甲基纤维素半固体培养基重悬细胞沉淀，通过优化甲基纤维素半固体培养基中甲基纤维素、血清及L-谷氨酰胺三种成分的浓度，建立稳定高效的杂交瘤细胞克隆化方法。并进行有限稀释法和甲基纤维素半固体培养基法筛选杂交瘤细胞的对照实验。阳性克隆扩大培养，ELISA检测后冻存，再次复苏后ELISA检测呈阳性为稳定杂交瘤细胞株。结果甲基纤维素三种浓度中，甲基纤维素浓度1．0％和1．2％时获得的杂交瘤细胞团个数约为甲基纤维素浓度1．25％时的2倍。而甲基纤维素浓度1．2％时细胞团固定效果较甲基纤维素浓度1．0％更好。通过对梯度稀释的SP 2／0细胞培养后，选择半固体培养基中使用的胎牛血清浓度（25％），在半固体培养基中含4mmol／L L-谷氨酰胺比含2mmol／L L-谷氨酰胺得到多近一倍的杂交瘤。得到的5株阳性杂交瘤在扩大培养、冻存复苏后抗体分泌稳定，得到5株稳定阳性杂交瘤细胞株。有限稀释法和甲基纤维素半固体培养基法筛选杂交瘤细胞对照实验显示半固体培养基法可节省一半时间。结论建立了稳定的甲基纤维素半固体培养基一步法筛选和克隆化杂交瘤细胞的方法。该法缩短了克隆化的时间，节省了人力和材料．不需要添加饲养细胞和细胞因子，是一种稳定高效的杂交瘤细胞克隆化方法。

Feasibility Study on the Use of Semi-solid Medium for the Preparation of Monoclonal Antibodies

Objective To develop a methylcellulose semi-solid medium based method for the selection and cloning of monoclonal antibodies. Methods After cell fusion, the cell pellet was resuspended in methylcellulose semi-solid medium. The concentrations of methylcellulose, serum and L-Glutamine were optimized to improve the stability and efficiency of the culture medium.The two methods of cloning of hybridoma by limiting dilution and methylcellulose semi-solid medium were compared.Positive clones were expanded and frozen.Results The number of hybridoma obtained from the 1.0%-1.2% methylcellulose medium was 2 times greater than the 1.25% medium.The growth of hybridoma in 1.2% methylcellulose medium was better than the 1.0% medium. After the culture of gradient dilution of SP 2/0,25% FBS was used in the medium.One times more clones were produced in the medium containing 4mmol/L L-Glutamine than that containing 2 mmol/L L-Glutamine.The positive clones were stable after recovery. Methylcellulose semi-solid medium method saved half of the time than limited dilution method. Conclusion A stable and efficient methylcellulose semi-solid medium based method was established for the cloning of hybridoma for the production of monoclonal antibodies.