| 袁媛1 徐瑞芬2 何争3 李成祥3 李静霞3 王海昌3 冯旭阳3.天然属性抗LDL 及oxLDL IgM 亚类抗体的制备与鉴定[J].,2011,11(6):1043-1047 |
| 天然属性抗LDL 及oxLDL IgM 亚类抗体的制备与鉴定 |
| Generation and Identification of Natural Monoclonal Antibodiesagainst LDL and oxLDL |
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| DOI： |
| 中文关键词: 天然抗体 单克隆抗体 杂交瘤技术 低密度脂蛋白 氧化低密度脂蛋白 |
| 英文关键词: Natural antibodies Monoclonal antibodies Hybridoma LDL oxLDL |
| 基金项目:国家自然科学基金资助（81070248） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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