摘要
基于 Western 印迹技术的基本原理,利用 IgE 结合因子/可溶性 CD23(IgEBF/sCD23)能够和 IgEFC 端特异性结合的特点,在系统研究了包括:IgE 浓度,反应时间及转移条件等数种因素后,首创了 IgE 介导的间接 Western 印迹技术.用于对 SDS—PAGE 电泳分离后样品中 IgEBF/sCD23特异性片段进行鉴定.实践证明:该法具有重复性高,操作简便及节约资金等优点.运用该技术,我们确定了 RPMI8866细胞上清中具有 IgEBF 活性的五种不同分子量的多肽.此方法的建立对开展 IgEBF/sCD23的研究以及其它特异性分子的鉴定都具有参考价值.
To detedt IgE binding fators/soluble CD23 (IgE BF/sCD23),an IgE mediated Western system was established on the basic priciple of Western blotting.In this system,polypeptides in the concentrated suspension of RPMI8866 cell llne culture were firstly separated with SDS-PAGE electrophoresis.Then,the separated components ere blotted onto nitrocellulose sheets with smei-dry electrophoretic blotting equipment.IgeBF/sCD23 was detected with their natural ligand-human myeloma IgE.The second antibodies(mouse anti-human IgE McAb or horse anti-human IgE)labled with horse radish peroxidase were applied to colour the specific bands.In this paper,the onditions,
including blotting time,concentrations of IgE and reaction time ect,were investigated.The advantages of this system are that the numbers of RPMI8866 required to produce enough IgEBF are greatly reduced and CD23 McAb is not necessary.With this system,5 bands(18KD,37KD,42KD,45KD and 86KD)with IgEBF activity were identified.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
1992年第3期169-173,共5页
Chinese Journal of Immunology
基金
国家七五重点攻关项目
青年自然科学基金
