目的建立一种敏感、特异、便捷的方法,用于检测食源性金黄色葡萄球菌肠毒素A(SEA)。方法采用经典的细胞融合技术制备抗SEA的单克隆抗体,亲和层析纯化抗体蛋白,ELISA方法测定抗体滴度,免疫层析分析抗体亚型。以抗SEA单抗为一抗,建立Western印迹法,检测不同食物样品中的SEA。结果筛选出3株抗SEA的单抗,命名为SEA-7,SEA-18和SEA-86。纯化后的3株单抗纯度均>90%,抗体滴度均在1∶32 000,经鉴定均为IgG1亚型。利用3株单抗建立检测SEA的Western印迹法,SEA-7的检测灵敏度最高,可达1.56 ng。以SEA-7为一抗,可检测出不同食物中污染的SEA,如污染了1.56 ng SEA的水,污染了5 ng SEA的粥或火腿肠匀浆。结论建立的检测SEA方法可靠灵敏,可用于监测食源性金黄色葡萄球菌污染。
Objective: The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initially in clinical immunoassay. Methods: Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by affinity chromatography. Antibody titer was determined by enzyme linked immunosorbent assay(ELISA). The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results: Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by affinity chromatography. The purity of antibodies was higher than90%. The titers of antibodies were more than 1: 6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor cell lines and determine the expression of P53 in tumor tissue. Conclusion: Three strains of monoclonal antibodies with high affinity to P53 were successfully established, which can be used for detecting the expression of P53 in tumor cells or tissue.
