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目的研制异硫氰酸荧光素(Fluoresceinisothiocyanate,FITC)标记的直标单克隆抗体2F9-FITC,用于白血病的免疫分型诊断。方法2F9杂交瘤细胞的亚克隆,2F9腹水的制备、纯化、纯度鉴定按照常规方法进行;参考改良Marsshall氏法,制备FITC标记的2F9-FITC单抗;通过流式细胞术鉴定单抗亚型,比较2F9-FITC和标准CD14-FITC在不同类型白血病细胞上的表达情况。结果制备了大量高纯度的IgG1κ亚型2F9单抗,成功研制了直标单抗2F9-FITC,A495/A280比值为0.44;2F9-FITC与CD14-FITC在单核细胞上反应的阳性细胞百分数分别为84.50%及90.08%,而在淋巴细胞仅为0.52%和1.01%;2F9-FITC与标准CD14-FITC在各类白血病细胞上的阳性细胞百分数的差异不显著(n=23,t=0.922,P=0.367)。结论研制的2F9-FITC能够替代进口的同类高质量单抗试剂,用于单核细胞相关性白血病的免疫诊断。
Objective To develop a direct monoclonal antibody labeled with fluorescein isothiocyanate (FITC), 2F9-FITC, for immunophenotyping of leukemia. Methods 2F9 hybridoma cells subclone, 2F9 ascites preparation, purification, purity identification carried out according to conventional methods; reference improved Marsshall’s method, FITC-labeled 2F9-FITC monoclonal antibody; identified by flow cytometry subtype of monoclonal antibody, The expression of 2F9-FITC and standard CD14-FITC on different types of leukemia cells was compared. Results A large number of IgG1 kappa subtype 2F9 McAbs were prepared and the direct monoclonal antibody 2F9-FITC was successfully developed. The A495 / A280 ratio was 0.44. The percentages of positive cells that reacted with 2F9-FITC and CD14-FITC on monocytes were FITC was 84.50% and 90.08%, respectively, but only 0.52% and 1.01% in lymphocytes. There was no significant difference in the percentage of positive cells between 2F9-FITC and standard CD14-FITC in all kinds of leukemia cells (n = 23, t = 0.922, P = 0.367). Conclusions The 2F9-FITC was developed to replace the imported high-quality monoclonal antibodies for the immunological diagnosis of monocyte-associated leukemia.