晚期氧化蛋白产物抗体的制备和鉴定.docVIP

晚期氧化蛋白产物抗体的制备和鉴定* 田梅1， 易维京2，吴雄飞1， 余荣杰1 (1 第三军医大学西南医院肾科, 2第三军医大学临床生化教研室，重庆 400038) 摘 要：目的 制备和鉴定抗人晚期氧化蛋白产物 （AOPPs）单克隆抗体 (McAb) 。方法 用次氯酸（HOCL）和纯化的人血清白蛋白(HSA)孵育获得 AOPPs-HSA，取凝胶层析纯化后的第 1峰 AOPPs-HSA 作为免疫原，免疫 BALB /c小鼠，采用杂交瘤技术制备抗 AOPPs-HSA 的 McAb，用间接 ELISA、western blot鉴定其特异性及亚类。结果 成功筛选出3株稳定分泌抗 AOPPs，1株既抗HSA又抗AOPP的 McAb杂交瘤细胞株，其分泌抗体亚类3株为IgG1，1株为IgG2b， 间接ELISA和Western blot证明制备的抗体可以特异地和天然或经HOCL与HSA处理后纯化的AOPPs结合。结论 获得抗人 AOPPs-HSA的 McAb细胞株 4株，为进一步研究 AOPPs生物学活性及 AOPPs相关疾病的诊断和预后奠定基础。 关键词：晚期氧化蛋白产物；单克隆抗体；制备和鉴定 Preparation and identity of monoclonal antibody for advanced oxidation protein products. TIAN Mei,YI Weijing,WU Xiongfei,YU Rongjie. (1Department of Kidney, Southwest Hospital, 2Department of Clinical Biochemistry, College of Medical Laboratory,Third Military Medical University,Chongqing 400038, China) Abstract: Objective To prepare and identify the monoclonal antibodies(McAb) against AOPPs. Methods BALB /c mice were immunized with purified AOPPs-HSA, McAbs against AOPPs-HSA was produced by hybridoma technique. Indirect ELISA and western blot were used to identify the immunoglobin specificity and subtype. Results Three novel McAbs only against AOPPs-HSA and one against AOPPs-HSA and HSA were harvested.The subclass of three McAbs were determined as IgG1 and one McAb as IgG2b. McAbs reacted strongly and specifically to protein of nature and HOCL-treated AOPPs by Western blot analysis. Conclusion The four McAbs against AOPPs prepared and identified successfully in this study may provide an important tool for studying on the biological properties of AOPPs and for its detection clinical laboratory. Key words：advanced oxidation protein products；onoclonal antibody； reparation and identification 晚期氧化蛋白产物(Advanced oxidation protein products，AOPPs)是Witko-Sarsat等于1996年首先报道的由尿毒症血液透析患者血浆中分离出的含有双酪氨酸的蛋白交联产物，系因患者体内过高的氧化应激水平导致各种蛋白质氧化损伤所形成的终末产物的总称[1]。是慢性肾功能衰竭 (Chronic renal failure，CRF)患者免疫功能紊乱、加速性动脉粥样硬化、透析相关性淀粉样变等长期并发症的重要致病环节[2]。目前的研究认为AOPPs还参与了多种生理及病理过程，如衰老、肥胖、老年性白内障、动脉粥