脂蛋白脂肪酶单克隆抗体的制备及特性

Mouse Monoclonal Antibodies against Bovine Milk Lipoprotein Lipase: Production and Characterization

为了研究脂蛋白脂肪酶的构效关系及其在动脉粥样硬化中的病理作用机制，用牛奶纯化的脂蛋白脂肪酸免疫Balb/c小鼠通过杂交瘤技术建立了30余株分泌抗牛脂蛋白脂肪酸单克隆抗体的杂交瘤细胞。对其中2E5、2G10、6D7、8D2、7F4进行特征分析发现：①其抗体类别均为IgG，亚类分别为IgG1、IgG2b和IgG2b、IgG1、IgG1；②抗体效价为5×10-2～2×10-3；③5种单克隆抗体可认为识别三个不同的抗原表位；④West－ern－blot及Dot－blot结果显示各单克隆抗体均能识别纯化的脂蛋白脂肪酸，与人奶均无交叉反应，与牛奶显示不同的结合特征。

Aim Studies were performed to produce specificmonoclonal antibodies to bovine milk lipoprotein lipase(bLPL), to verify the specificity of the monoclonal an-tibodies for bLPL, and to characterize them.Metkods Purified lipoprotein lipase (LPL) frombovine milk-56 kDa was used as an antigen for the pro-duction of anti-LPL antibodies in mice. The spleen wasremoved from the animal having the highest titer of an-tibodies to LPL and the cells were fused with mousemyeloma cells. After cultured with hypoxanthine-aminopterin-thymidine (HAT ) culture medium, thisprocedure thus identified only those hybridomas whichproduced antibodies directed against LPL for super-natants by enzyme linked immunosorbent assay(ELISA). After cloning five monoclonal cell strainswere established, named as 2E5, 2G10, 6D7, 8D2 and7F4. The titriton of monoclonal antibodies (McAb) are5 × 10-2~2 × 10-3 for supernatants by ELISA.Results and Conclusion Contitration study showedMcAb recognized three different epitopes of LPL: 6D7and 8D2 to a close epitop; 2G10 and 7F4 to other closeepitope; 2G10 but recognized a far epitope away fromthe two of others. Western blot analysis of bovine milkwith the McAb gave a single protein band of 56 kDa;2G10 gave two protein bands of 50 kDa and 47 kDa.Dot-blot analysis of bovine milk with the McAb 2E5,8D2 and 6D7 showed specific reaction Western blot andDet-blot analysis of human milk with the McAb, hadno cross-reacted.