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采用辛酸-硫酸铵沉淀法从大鼠杂交瘤腹水中纯化大鼠抗人肝癌BEL-7402细胞单克隆抗体3A5,纯度大于90%,抗体类型为IgGI型,与人肝癌BEL-7402细胞及小鼠结肠癌C-26细胞均有强免疫反应性.为探讨Fab’片段制备方法,首先分别以胃蛋白酶、菠萝蛋白酶和无花果蛋白酶消化3A5,结果表明3种酶均可使3A5酶解产生F(ab’)_2,其中以胃蛋白酶法在合适的反应条件下产生的F(ab’)_2产率较高,与BEL-7402细胞的免疫反应性为完整抗体的60%.在此基础上,以二巯基苏糖醇(DTT)还原F(ab’)_2并以DE-52纯化可以得到保留了抗体绞链区巯基的Fab’片段,可用于药物的定向偶联,其与BEL-74O2细胞的免疫反应性约为完整抗体的20%.以葡聚糖T-40(DextranT-40)为中介体,将Fab’与抗肿瘤抗生素平阳霉素相偶联,制得偶联物.偶联分子比为1:50.氯代三苯基四氟唑(TTC)抑菌效价检测法测定偶联物中平阳霉素
Rat anti-human hepatocellular carcinoma BEL-7402 cell monoclonal antibody 3A5 was purified from the rat hybridomas ascites by precipitation with ammonium octanoate-ammonium sulphate. The purity was greater than 90%, the antibody type was IgGI, and the human hepatocellular carcinoma BEL-7402 cells and mice Colon cancer C-26 cells all have strong immunoreactivity. To investigate the preparation method of Fab’ fragments, 3A5 was first digested with pepsin, bromelain and ficin. The results showed that all three enzymes can make 3A5 enzymatic hydrolysis to generate F(ab). ’)_2, wherein the yield of F(ab’)_2 produced by pepsin under suitable reaction conditions is high, and the immunoreactivity with BEL-7402 cells is 60% of intact antibody. On this basis, Dithiothreitol (DTT) reduces F(ab’)_2 and purifies with DE-52 to obtain the Fab’ fragment which retains the sulfhydryl group in the antibody hinge region. It can be used for the directional coupling of drugs, and it can be used for the BEL-74O2 cells. The immunoreactivity is about 20% of the intact antibody. Using Dextran T-40 as the mediator, Fab’ is coupled with the antitumor antibiotic antibiotics pingyangmycin to prepare conjugates. A ratio of 1:50. Chlorotriphenyltetrazolium (TTC) bacteriostatic titer assay for the determination of conjugates in pingyangmycin