利用改良后的脾内免疫和半固体培养基法制备单克隆抗体

The Improved Intrasplenic Immunization and Semi-solid Medium to Prepare Monoclonal Antibody

旨在利用改良后的脾内免疫和半固体培养基法制备单克隆抗体,节约免疫原用量、简化试验操作、一步法筛选和克隆杂交瘤细胞、缩短单抗制备周期。通过局部麻醉小鼠脾脏区域皮肤,切开后透过腹膜直接向脾脏内注射微量抗原,使小鼠的脾细胞在短时间内产生免疫反应,尾静脉注射强化免疫后进行细胞融合;选取半固体培养基(甲基纤维素、软琼脂)法,以有限稀释法作对照筛选分泌高效价抗体的杂交瘤细胞。结果显示,与传统的单抗制备方法相比,改进后的脾内免疫操作更简单,联合尾静脉注射,使用微量免疫原在短时间内产生高效价抗体;半固体培养基甲基纤维素浓度在1.1%条件下,获得克隆团较多,阳性率较高。本研究方法适用于大规模制备单克隆抗体。

It was to utilize the improved intrasplenic immunization and semi-solid culture medium to reduce the immune dosage,clone the hybridoma simply,and shorten the period of cycle.The skin areas of mice spleen was anesthetized,then the spleen was injected with trace antigen,which could be immuned in a short period of time,cell fusion was prepared after intravenous injection.The three different methods（limited dilution,methylcellulose,soft agar）were used to get hybridomas which can secret monoclonal antibody.Results showed that compared with the traditional method of preparation of monoclonal antibody,the improved intrasplenic immunization was simpler operation,without the addition of adjuvant,and the high titer of antibody was getted in a short time.Positive rate was the highest when methylcellulose concentration in the 1.1% condition.It was suggested that these methods could be suitable for large scale preparation of monoclonal antibody.