糖化酶单抗的研制及其抗原识别位点的分析  

The Development and Antigenic Analysis of Monoclonal Antibodies to Glucoamylase

作　　者：张丹[1] 姜威[1] 李双喜[1] 汪惠泽[1] 冯云飞[1] 刘云迎[1] 李东明 何祥来[3] 王捍东[1] 

机构地区：[1]扬州大学兽医学院 江苏省普通高校重点学科内科实验室,江苏扬州225009 [2]江苏省常州市出入境检验检疫局,江苏常州213022 [3]江苏畜牧兽医职业技术学院,江苏泰州225300

出　　处：《中国畜牧兽医》2012年第11期39-42,共4页China Animal Husbandry & Veterinary Medicine

基　　金：江苏高校优势学科建设工程和江苏省高校重点实验室开放课题资助

摘　　要：为建立掺糖造假蜂蜜中残留糖化酶的检测方法,用从黑曲霉中提取的糖化酶作为免疫原,免疫BALB/c小鼠,利用杂交瘤技术获得了12株稳定分泌针对糖化酶抗体的杂交瘤细胞株。单克隆抗体亚型鉴定结果显示,10株为IgG1,2株为IgG2b,轻链均为κ轻链。Western blotting分析结果表明,12株抗体均可特异性结合糖化酶。其中6株单抗(McAb-2H4F9、6H9D8、8F2F11、8F2E9、1A8G6、1C4D5)细胞株采用体内诱生法制备的腹水效价均1∶1×104以上。采用抗体叠加试验对这6株抗糖化酶单抗的抗原识别位点进行检测,反应增殖结果表明,6株单抗分别针对4类不同抗原位点,McAb-6H9D8和McAb-8F2F11针对第Ⅰ种抗原决定簇;McAb-1A8G6和McAb-1C4D5针对第Ⅱ种抗原决定簇;McAb-8F2E9针对第Ⅲ种抗原决定簇;McAb-2H4F9针对第Ⅳ种抗原决定簇。制备的抗体针对不同的抗原表位,为双抗夹心ELISA方法的建立提供前提。To develop a detection method for glucoamylase in honey,BALB/c mice were immunized with the glucoamylase from Aspergillus niger.Splenocytes of the immunized mice were fused with SP2/0 cells with PEG.12 monoclonal antibodies（McAbs） against the glucoamylase were obtained and identified.10 hybridoma cell lines represented subtypes IgG1 and the other were IgG2b,and their light chains were κ chain.Western blotting analysis showed that the 12 McAbs specifically recognized glucoamylase.Hybridoma 2H4F9,6H9D8,8F2F11,8F2E9,1A8G6,1C4D5 excreting anti-glucoamylase McAbs were used to be injected into abdominal cavity of mice（ICR）.The titers of the McAbs were all above 1∶1×104.Six strains of monoclonal antibodies were confirmed to be specific for four different kinds of epitopes on glucoamylase competitive binding assay.Among them,McAb-6H9D8 and McAb-8F2F11 might direct to the same antigenic determinant on the GA,McAb-1A8G6 and McAb-1C4D5 to the second antigenic site,McAb-8F2E9 to the third antigenic site,McAb-2H4F9 to the fourth antigenic site.

关 键 词：淀粉糖化酶 单克隆抗体 抗原识别位点 

分 类 号：S852.43[农业科学—基础兽医学]