基于单克隆抗体的三聚氰胺ELISA检测方法的建立

Establishiment of ELISA Methods for Detecting Melamine Based on Monoclonal Antibodies

DOI：

中文关键词: 三聚氰胺 单克隆抗体 间接竞争ELISA 直接竞争ELISA

英文关键词: melamine monoclonal antibody direct competitive ELISA indirect competitive ELISA

基金项目:

摘要点击次数: 189

全文下载次数: 655

中文摘要:

建立三聚氰胺直接和间接竞争ELISA检测方法。三聚氰胺(melamine)-BSA免疫BALB/c小鼠,通过融合小鼠脾细胞与SP2/0细胞,制备分泌MEL单克隆抗体(monoclonal anti-body)的细胞株;体内诱生腹水制备MEL-mAb。用高碘酸钠法制备MEL-OVA-HRP偶联物并对其活性进行鉴定。分别用MEL-OVA和制备的MEL-mAb包被,用三聚氰胺标准液作为抑制物,做直接和间接竞争ELISA,并对两种ELISA方法在灵敏度、线性检测范围、检测时间进行比较。筛选出4株可以稳定分泌特异性抗体的杂交瘤细胞株。其中的C4株腹水效价达到1∶104,亲和力常数(Ka)为2.92×108L/mol。三聚氰胺间接竞争ELISA和直接竞争ELISA检测法的IC50分别为3.12μg/L和56.88μg/L,线性检测范围分别为0.05～10μg/L和0.1～1 000μg/L。两种竞争ELISA方法都能应用于三聚氰胺的快速检测,但间接竞争ELISA比直接竞争ELISA更加灵敏。

英文摘要:

The objectiveof this study is to establish direct and indirect competitive enzyme-linked immunoassay for detection of melamine.For this,BALB/c mice were immunized with MEL-BSA,after a fusion of mouse spleen cells with SP2/0 cells,the hybridoma cells secreting monoclonal antibody against MEL were generated.MEL-mAb was produced by ascites in mice.Melamine was conjugated with HRP,and the conjugates activity was identified.MEL-OVA and MEL mAb were coated in wells respectively,standard melamine were used as inhibitors,direct and indirect competitive ELISA were compared in sensitivity,work range and testing time.Four hybridism cell strains were obtained,which could stably secrete mAbs against melamine.One of them,C4 strain,had a titers of 1∶104 at least in ascites by indirect ELISA and a high affinity constant(Ka) with 2.92×108L/mol.IC50 of direct and indirect competitive ELISA methods were 3.12 μg/L and 56.88 μg/L respectively,and their work ranges were 0.1~1000 μg/L and 0.05~10 μg/L,respectively.Both direct and indirect competitive ELISA methods can be used for the detection of melamine,while the latter is more sensitive and suitable for further research.

查看全文 查看/发表评论 下载PDF阅读器