天然属性抗LDL及oxLDL IgM亚类抗体的制备与鉴定

目的：制备天然属性抗低密度脂蛋白（LDL）及抗氧化低密度脂蛋白（oxLDL）IgM亚类抗体。方法：给予Babl/c小鼠高胆固醇饮食,4周后取脾细胞直接与SP2/0细胞融合,以纯化的LDL及oxLDL为抗原,对阳性杂交瘤细胞生长孔进行间接ELISA筛选。鉴定杂交瘤上清的免疫球蛋白亚类,进而采用免疫沉淀和免疫印迹法对获得的抗体进行免疫学反应性鉴定。结果：杂交瘤细胞分泌的抗LDL及抗oxLDL的天然抗体通过ELISA法被筛选出来,可以与LDL或oxLDL发生高亲和力结合,经过4次克隆化,最终获得2株稳定分泌天然抗LDL的抗体,命名为5G8和2H7,及1株稳定抗oxLDL的抗体,命名为3A6,3株抗体均属于IgM亚类,无交叉反应,可以满足免疫印迹、免疫沉淀等实验要求。结论：成功制备了抗LDL及抗oxLDL IgM亚类抗体,为研究天然抗体在体内脂质代谢和相关心脑血管疾病如动脉粥样硬化等发生发展中的作用提供了重要的研究工具。

Generation and Identification of Natural Monoclonal Antibodies against LDL and oxLDL

Objective：To produce natural mouse IgM monoclonal antibodies（MAbs） against low-density lipoprotein and oxidized low-density lipoprotein with high specificity and activity.Methods：BALB/c mice were fed with high cholesterol diet;The splenocytes cells were directly fused with Sp2/0 myeloma cells by standard hybridoma production techniques.Hybridomas were selected on the basis of the ability of supernatant to bind natural LDL and oxLDL in indirect ELISA.Hybridomas were identified by western blotting and immunoprecipitation.Results：The hybridomas producing anti-LDL and anti-oxLDL antibodies were screened by enzyme-linked immunosorbent assay（ELISA） and isotype was identified.Two hybridoma cell lines,named 5G8,and 2H7,were developed which could secrete anti-LDL MAbs stably.One hybridoma cell lines,named 3A6 was developed which could secrete anti-oxLDL MAbs stably.The three hybridoma cell lines were all belonged to IgM subclass,and no cross reactions were found between these MAbs.The specificity of MAb was determined based on activity of western blotting and immunoprecipitation.Conclusion：The natural mouse IgM monoclonal antibodies（MAbs） against LDL and against oxLDL with high specificity and activity were produced successfully by using standard hybridoma production techniques,which could provide potential tool for the research on lipid metabolism and atherosclerosis progression.