抗紫杉醇单克隆抗体的制备及ELISA检测方法的建立  

Preparation of monoclonal antibody against taxol and establishment of ELlSA detection method

作　　者：赵凯[1,2] 孙立新[3] 孙宇石[1] 周东坡[1,2] 

机构地区：[1]黑龙江大学生命科学学院,微生物黑龙江省高校重点实验室,哈尔滨150080 [2]农业微生物技术教育部工程研究中心,哈尔滨150500 [3]黑龙江大学校医院,哈尔滨150080

出　　处：《中国新药杂志》2012年第4期436-442,共7页Chinese Journal of New Drugs

基　　金：国家自然科学基金(30970090);黑龙江省普通高等学校新世纪优秀人才培养计划(1251-NCET-005);哈尔滨市科技创新人才研究专项基金(2010RFQXS043);黑龙江大学高层次人才(创新团队)支持计划(Hdtd 2010-17)

摘　　要：目的:制备抗紫杉醇单克隆抗体并建立间接ELISA检测方法。方法:用琥珀酸酐法将紫杉醇(taxol)与牛血清白蛋白(BSA)偶联制成全抗原taxol-BSA,采用紫外波长扫描法和薄层层析检测法对合成抗原进行鉴定;以taxol-BSA免疫Blab/c小鼠,取免疫小鼠脾细胞与SP2/0进行融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等筛选出单克隆抗体杂交瘤细胞株,并对单克隆抗体的特异性进行鉴定;用杂交瘤细胞株诱生小鼠腹水,应用辛酸-饱和硫酸铵沉淀法纯化腹水。同时,对间接ELISA法反应条件进行了优化。结果:获得了3株能稳定分泌抗紫杉醇单克隆抗体杂交瘤细胞株,分别命名为taxol-A1,taxol-A2和taxol-A3;taxol-A3株腹水效价为1∶128 000,属于IgG2b亚型;建立的间接ELISA法检测条件为15μg.mL-1抗原、4℃包被过夜、1%BSA封闭1 h,PBS做抗体稀释液,单抗反应时间为30 min,酶标二抗作用时间1 h,10%浓硫酸终止反应。结论:获得了特异性的抗紫杉醇单克隆抗体并建立了间接ELISA检测方法。本研究制备的抗紫杉醇单克隆抗体及建立的间接ELISA检测方法,为从内生真菌生物合成紫杉醇发酵液中迅速、高效地分离纯化和检测紫杉醇奠定了基础。Objective： To prepare monoclonal antibodies（McAb） against taxol and to establish an indirect enzyme-linked immunosordent assay（ELISA） method for rapidly detecting taxol.Methods： Taxol was coupled with the carrier protein bovine serum albumin（BSA） to prepare the complete antigen taxol-BSA by succinct anhydride method.The prepared taxol-BSA was identified by ultraviolet scanner and thin layer chromatography（TLC）.Blab/c mice were immunized with taxol-BSA.The splenocytes of the immunized Blab/c mice were fused with SP2/0 myeloma cells by PEG 4000,and selected with HAT medium.Antibodies to taxol were screened by indirect ELISA.The hybridoma cells that secrete high affinity monoclonal antibodies against taxol were cloned by a limiting dilution method.Meanwhile,the conditions of indirect ELISA for determination of antibody against taxol were optimized.Results： Three hybridoma cell lines secreting the antibody,taxol-A1,taxol-A2 and taxol-A3,were obtained and the antibodies had been purified from ascites using caprylic acid/ammonium sulfate precipitation（CA-AS）.Subclasses of the purified antibodies belonged to IgG2b.The titer of antibody from the hybridoma cell taxol-A3-induced ascites was 1∶128000.The optimal detection conditions were 15μg·mL-1 of antigen in coating buffer,at 4℃,overnight,1% BSA blocking for 1h,PBS as dilution solution,30min of McAb reaction time,1h of enzyme-labeled antibody reaction time,and termination of reaction with 10% H2SO4.Conclusion： The monoclonal antibodies against taxol have been prepared,and the indirect ELISA method for rapidly detecting taxol is successfully established.This method provided an important basis for the detection of taxol produced by microbe fermentation in the future.

关 键 词：紫杉醇 内生真菌 taxol-BSA 抗紫杉醇单克隆抗体 酶联免疫吸附试验 

分 类 号：R979.1[医药卫生—药品]