海洋硫酸多糖类药物聚甘古酯单克隆抗体的制备及其特性研究  

Preparation and characterization of monoclonal antibody against marine sulfated polysaccharide drug poly-mannaguluronic acid

作　　者：陈騉[1] 辛现良[1] 耿美玉[1] 朱建春[2] 杨明[2] 李勇[2] 

机构地区：[1]青岛海洋大学药物与食品研究所,山东青岛266003 [2]长春生物制品研究所血液制品研究室,吉林长春130012

出　　处：《药学学报》2003年第1期23-26,共4页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金重点项目 (3 0 13 0 2 0 0 ) ;国家自然科学基金资助项目 (3 0 0 70 0 694)

摘　　要：目的　制备和纯化聚甘古酯 (911)单克隆抗体 ,为 911药代动力学的免疫学方法的建立提供依据。方法用还原胺化法 ,制备 911 BSA和 911 HSA复合物 ,间接ELISA法检测抗体生成。以IsostripTM 试剂盒测定抗体亚型 ,流式细胞仪测定DNA含量。结果　获得了一株阳性杂交瘤细胞DD3,腹水效价可达 1× 10 5,其抗体亚型为IgG2a(κ) ,细胞株的DNA含量约为脾细胞和NS 1细胞之和 ,亲和力常数为 2 0× 10 8L·mol- 1 。结论　本实验制备了特异性针对 911的单克隆抗体DD3,且与内源性多糖和褐藻酸无交叉反应 ,为 911药代动力学的免疫学检测提供了依据。Aim To prepare and characterize the monoclonal antibody against poly-mannaguluronic acid (911). Methods A hybridoma cell line was obtained by cloning for 3 times those cells that secret antibody against 911 after the fusion of NS-1 myelome cells with spleen cells from Balb/c mice immunized with a 911-bovine serum albumin conjugate, prepared by reductive amination. Hybridoma was inoculated to Balb/c mouse to induce ascites. The antibody was purified by ammonium sulfate and Protein A - Sepharose CL-4B. DD3 was confirmed to be fusion cells after determination of DNA content with flow cytometry. Competitive inhibitory test and Biacore confirmed the cross-reactivity of the antibody with other endogenous polysaccharides or with alginic acid sodium. Igs′ classes and subclasses were identified by Isostrip TM method. The affinity of DD3 was verified by ELISA. Results A hybridoma cell line secreting monoclonal antibody against 911 (marine sulfate polysaccharide) named DD3 was obtained. The DD3 ascites contained specific antibody with titer over 1.0×10 5. There was no cross-reactivity of these antibodies with other endogenous polysaccharides or with alginic acid sodium. The immunoglobulin subclass of DD3 was IgG2a, κ type. The affinity of DD3 was 2.0×10 8 L·mol -1 . Conclusion One hybirdoma line (DD3) secreting monoclonal antibody against 911 was established to provide a potential method for the pharmacokinetic study of 911.

关 键 词：海洋硫酸多糖 聚甘古酯 单克隆抗体 抗艾滋病药物 药代动力学 免疫学 

分 类 号：R967[医药卫生—药理学] R978.7[医药卫生—药学]