摘要
目的制备高特异性的小鼠抗人IgE单克隆抗体(mAb)作为候选免疫吸附剂。方法用重组人IgE重链2~4区(IgECε2-4)免疫BALB/c小鼠,经细胞融合后,用甲基纤维素半固体培养基法筛选杂交瘤细胞。用IgECε2-4包被酶标板,间接ELISA筛选阳性细胞株,用天然IgE、IgG、IgM、IgA、IgD包被酶标板,间接ELISA检测阳性杂交瘤细胞无血清培养上清与天然IgE的亲和性和特异性;将稳定分泌特异性抗IgE mAb的细胞株扩大培养,小鼠体内诱生腹水法制备mAb,检测腹水的效价和亲和力,利用mAb亚型鉴定试剂盒检测获得的mAb的亚型。结果经过5次细胞融合总共得到29株阳性杂交瘤细胞,有11株与天然IgE亲和性较强,有2株与其他免疫球蛋白几乎无交叉反应,分别为3E9和7B4。结论成功制备了小鼠抗人IgE mAb,该mAb具有高效价、较高亲和力和高特异性。
Objective To prepare the high-affinity and high-specificity mouse anti-human IgE monoclonal antibody(mAb)as a candidate immunosorbent.Methods BALB/c mice were immunized using recombinant antigen IgECε2-4.Coated ELISA plate with IgECε2-4 was used to screen positive cell lines by indirect ELISA,then coated ELISA plate with natural IgE,IgG,IgM,IgA,IgD to detect the affinity and specificity of serum-free culture supernatant of positive hybridoma cells with natural IgE.The cell line stably secreting specific anti-IgE monoclonal antibodies was expanded and inoculated into the abdominal cavity of BALB/c mice to prepare mAbs.The secreted mAbs were identified by ELISA kit followed by the identification of mAb subtypes.Results The 29 positive hybridoma cells were obtained after five cell fusions,of which 11 strains had strong affinity with natural IgE and 2 strains did not cross-react with other immunoglobulins 3E9 and 7B4.Conclusion The study successfully prepared mAbs against human IgE with high titer,affinity and specificity.
作者
续艳梅
杨正根
刘诗俐
黄慧慧
董文其
XU Yanmei;YANG Zhenggen;LIU Shili;HUANG Huihui;DONG Wenqi(School of Laboratory Medicine and Biotechnology,Southern Medical University,Guangzhou 510000;Guangzhou Koncen Bio Science Co.,Ltd,Guangzhou 510000,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2020年第7期640-644,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
产学研协同创新重大专项(201604046010)。
