摘要
目的制备出高效价的抗重组志贺毒素ⅡB亚基(rStx2B)单克隆抗体。方法以融合蛋白EspA-Stx2B作为抗原,免疫Balb/c小鼠,以未免疫的Balb/c小鼠脾细胞为饲养细胞;运用细胞杂交瘤技术制备,运用间接ELISA法筛选产生针对rStx2B的单克隆抗体细胞株;体内诱生法产生腹水,饱和硫酸铵沉淀法初步纯化,再采用HiTraprProteinA柱进一步纯化,快速定性试纸鉴定McAb的Ig亚型,采用间接ELISA法相加实验鉴定抗原识别表位。结果获得3株产生针对rStx2B的单克隆抗体细胞株Stx2B-1H9、Stx2B-1H10、Stx2B-3E5,Ig亚型分别为IgG1κ型,IgG2aκ型,IgG2bκ型,亲和力常数分别为7.36×108、6.94×108、5.85×108。结论成功制备3株稳定分泌抗rStx2B的杂交瘤细胞株,产生的McAb特异性好,亲和力高,为应用这些单克隆抗体建立免疫亲和层析法纯化天然志贺毒素Ⅱ,鉴定Stx2B的表位,研究针对EHECO157:H7感染的治疗性抗体奠定了基础。
Objective To prepare high effective monoclonal antibodies against recombinant shiga toxin ⅡB subunit （rStx2B）. Methods Balb/c mice were immunized with EspA-Stx2B and the spleen cells of unimmunized Balb/c mice were used as feeder cells. Hybridoma cells lines of monoclonal antibodies against rStx2B were prepared by hybridoma technique, and then screened by ELISA. The ascites developed by injecting the hybridoma cells into abdominal cavity of Balb/c mice were sedimented by saturated ammonium sulfate （SAS）, and then purified with HiTrap rProtein A. The subclasses and isotypes of the induced antibody were identified by mouse monoclonal antibody isotyping Kit. The antigenic epitopes of the induced antibody were analyzed by the ELISA addiitivity test. Results Three hybridoma cell fines were obtained and named Stx2B-1Hg, Stx2B-1H10, and Stx2B-3E5, which could produce specific monoclonal antibodies against rStx2B. The isotypes of monoclonal antibodies were IgG1 k, IgG2ak, and IgG2bk with the affinity constants of 7.36×10^8, 6.94 ×10^8, and 5.85×10^8, respectively. Con- dutdon Three hybridoma cell fines are prepared successfully, which can secrete high-titer and high-speeific moneclonal antibodies against rStx2B. The results provide a basis for founding the method of immunoaflinity chromatography to purify the natural shiga toxin Ⅱ , accrediting the epitope of Stx2B, and developing the therapy antibody against infection of EHEC O157：H7.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第2期148-151,共4页
Immunological Journal
基金
军队"十一五"攻关项目(06G078)
国家"863"计划课题(2006AA02Z405)
