MTT比色法和ELISA方法检测重组人rhM-CSF的活性  

THE MTT COLORIMETRIC AND ELISA METHODS FOR THE ACTIVITIES ASSAY OF THE RECOMBINANT HUMAN MACROPHAGE COLONY STIMULATING FACTOR(rhM CSF)

作　　者：朱洁[1] 秦浚川[1] 张冬梅[1] 焦瑞清[1] 臧宇辉[1] 

机构地区：[1]南京大学生命科学院医药生物技术国家重点实验室,江苏南京210093

出　　处：《南京大学学报（自然科学版）》2000年第2期202-207,共6页Journal of Nanjing University（Natural Science）

基　　金：国家 8 6 3高技术发展项目! (No :86 3- 10 2 - 11- 8)

摘　　要：应用家蚕幼虫表达的重组人巨噬细胞集落刺激因子 (rhM CSF)为材料 ,探索了MTT比色法和ELISA方法测定rhM CSF活性的最佳实验条件 ,并将这两种方法测定rhM CSF精品、粗品和原料的结果与软琼脂集落刺激法所得结果进行了比较 .结果表明 :MTT比色法灵敏度较高 ,结果稳定可靠 ,周期较短 ,可用于批量检测和精确定量 .ELISA方法简便快捷 ,特异性高 ,适用于大批量检测 .可根据不同检测要求选择不同的检测方法 ,为rhM CSF的质量检控和临床分析研究提供了较为切实可行的检测手段 .The macrophage colony stimulating factor(rhM CSF) is a homodimeric glycoprotein regulating the survival, proliferation and differentiation of monocytes and macrophages. As a new medicine of anti fungi and anti tumor,the rhM CSF may have a wider therapeutic window. The colony forming method is the earliest and an universal activities assay method of rhM CSF,but its use in a large number of quantitative assays has been limited due to several factors, such as taking much time,the complexity of process and unaufomafic. To monitor the preparation of the recombinant human macrophage colony stimulating factor expressed in silkworm larvae, we developed 2 methods for the activities assay of rhM CSF: one is the MTT colorimetric method detecting the activities of rhM CSF on BMM(Bone Marrow Derived Macrophage) cells,which were induced from the C 57 mouse's bone marrow cells(BMC) by incubation with 1?000 CFU/mL rhM CSF;and the other is ELISA method using the monoclonal antibody of rhM CSF to detect rhM CSF.The optimum experimental conditions were studied to increase the sensitivity.The optimum density of BMM cells is 6×10 5 cells/mL,and the optimum incubation time is 96 hours in the MTT method.The optimal concentration of monoclonal antibody of rhM CSF is 300 ng/mL in the ELISA method.Besides the purified,the semi purified and the crude samples were detected in the MTT and ELISA methods. The results obtained from these 2 methods are compared with those obtained from the colony forming method.Several phenomena emerged from this comparison. First,the sensitivity of the MTT method was higher than those of the other two in the purified samples' detection.Secondly,the activity of the semi purified and the crude samples detected in the MTT method was lower than that from the colony forming method.This indicated that there was something unknown restraining the proliferation of the BMM cells in unpurified samples.Finally,the results of different samples detected in the ELISA method were lower than the other t

关 键 词：ELISA MTT rhM-CSF 比色法 活性 基因工程 

分 类 号：Q78[生物学—分子生物学]