摘要
目的:制备鼠抗人CD28分子功能性单克隆抗体,研究其对T细胞活化、增殖及信号转导等方面的生物学效应。方法:以天然高表达CD28分子的人多发性骨髓瘤细胞株U266和小鼠淋巴瘤细胞转人CD28基因细胞株CD28-T分别作为免疫原和检测细胞株,采用B淋巴细胞杂交瘤技术进行单抗的研制;以快速定性试纸法鉴定单抗亚类;腹水诱生法和免疫亲和层析法进行单抗的制备和纯化;经间接免疫荧光法分析单抗对不同细胞膜表面CD28分子的识别;采用竞争抑制法分析单抗识别的抗原位点;利用3H-TdR掺入法分析单抗对PBTC的刺激效应和免疫荧光法分析PBTC活化前后的表型变化。结果:成功获得5株鼠抗人CD28功能性单克隆抗体,分别命名为2D5、2F5、3B6、3F8和8G8,其中2D5为IgG2a亚类,其余均为IgG1亚类;流式细胞仪分析结果显示,5株单抗均能良好识别CD28-T、U266、XG1和Jurkat细胞表面的CD28分子;竞争抑制试验表明,2D5和8G8能完全阻断标准抗人CD28单抗与U266膜CD28分子的结合,其余3株为部分阻断;3H-TdR掺入法实验结果表明,单抗8G8联合激发型CD3单抗能明显促进PBTC的增殖,刺激指数为7.4,活化细胞CD4、CD25、ICOS、41BB及OX40分子的表达上调。结论:5株单抗均为抗人CD28单克隆抗体,具有重要的基础研究及潜在的临床应用价值。
Objective:To prepare the monoclonal antibodies against human CD28 and study their biological effects.Methods:The hybridoma cell lines were obtained by using the B lymphoma technique after immunization of BALB/c mice with U266 expressing highly CD28, while CD28-T,transfected with full-length human CD28 cDNA.was served as screening cell lines. Recognition epitopes of mAbs were verified by competitive inhibitory test. Reactivites of mAbs to U266,CD28-T,XG1,Jurkat and Daudi cells were studied by indirect immunofluorescence assay.The mAb 8G8-inducing proliferation of peripheral T blood cells(PBTC) was determined by 3H-TdR incorporation,and phenotypic analysis of activated PBTC was studied by FACS.Results:Five mAbs(2D5,2F5,3B6,3F8,8G8) against CD28 were obtained. Subclass of 2D5 was IgG2a,while others all were IgG1. Each of five mAbs could react to CD28 molecules on CD28-T,U266,XG1 and Jurkat cells.2D5 and 8G8 recognized epitopes similar to standard anti-CD28 mAb.8G8 could promote proliferation of PBTC in vitro(SI=7.4),and could upregulate expression of CD4,CD25,ICOS,41BB and OX40 on PBTC. Conclusion: Five mAbs against CD28 may be of significant value in basic studies and clinic application.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2005年第6期406-410,共5页
Chinese Journal of Immunology
基金
本课题受国家重大基础研究项目资助(2001CB51003)
