摘要
本研究旨在制备针对白血病干细胞表面标志蛋白IL1RAP(IL-1 receptor accessory protein)的特异性单克隆抗体并对其进行鉴定。用重组人IL1RAP作为抗原免疫BALB/c小鼠,通过常规杂交瘤技术将免疫后的小鼠脾细胞与SP2/0细胞经PEG融合,筛选阳性杂交瘤细胞株。分别应用ELISA和Western blot法对杂交瘤细胞上清中分泌抗体进行抗体类型、滴度和敏感性检测。分离人外周单个核细胞,检测所得抗体对细胞内源性抗原的特异性。结果表明,获得了8株可稳定分泌IL1RAP单克隆抗体的杂交瘤细胞株,分别为3H6E10、4B6A6、8G11B5、9E9F2、10D8A7、1C7H7、1D7G11和2D3D3;抗体类型鉴定为IgG1/κ型;分泌的单克隆抗体能特异性识别单个核细胞表达的IL1RAP。结论:本实验成功制备了可稳定分泌IL1RAP单克隆抗体的杂交瘤细胞及其特异性的IL1RAP单克隆抗体,为将来有效清除体内白血病干细胞提供了新途径。
This study was aimed to prepare and identify human monoclonal antibody against IL-1 receptor accessory protein （IL1RAP）, which is a new identified surface marker for leukemia stem cell （LSC）, BALB/c mice were immu- nized with recombinant hu-IL1 RAP and the spleen ceils from immunized mice were fused with SP2/0 myeloma cells by conventional hybridoma technique. Positive hybridoma cells were selected and cultured. ELISA and Western blot were used to detect the type, titer and sensitivity of antibody. Peripheral blood mononuclear cells were isolated and used to test the antibody specificity. The results showed that 8 hybridoma cell lines able to stably secrete IL1RAP monoclonal anti- bodies were obtained and named 3H6E10,4B6A6,8G11 B5,9EgF2,10D8A7,1C7H7,1D7G11 and 2D3D3 respectively. These monoclonal antibodies belonging to IgG1/K type could specifically bind to IL1RAP from peripheral blood mononu- clear cells. It is concluded that the hybridoma cell lines with stable secretion of IL1RAP monoclonal antibodies is suc- cessfully constructed, thus providing novel ways to effectively clear LSC in the future.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2013年第6期1390-1393,共4页
Journal of Experimental Hematology
基金
国家自然科学基金(编号81200376
81270637)
中国博士后科学基金资助项目(编号2012M510141)
江苏省"六大人才高峰"资助(编号2012-WSN-082)
徐州医学院院长专项人才基金(编号2012KJZ13)
