【摘要】 目的　用于提取高纯度凝血因子X(FX)和制备缺乏FX血浆 ,以及建立FX抗原的ELISA检测方法等 .方法　用较高纯度的FX免疫Balb/C小鼠 ,经过 2次基础免疫 ,1次加强免疫 ,3d后取其脾细胞与骨髓瘤SP2 /0 Ag14细胞在PEG作用下进行细胞融合 ,经间接ELISA法筛选杂交瘤细胞株 ,通过免疫印迹试验、交叉反应试验、相加试验和二价金属离子依赖性试验等 ,对单克隆抗体进行鉴定 .结果　经 3～ 4次克隆化后获得了 10株能稳定分泌FX单克隆抗体 (FXMcAb)的杂交瘤细胞株 ,Ig亚类均为IgG1型 ,小鼠腹水效价均在 10 6以上 ,FXMcAb只与FX抗原起特异性反应 ,FXMcAb与FX抗原的结合不同程度地依赖于二价金属离子Zn2 + .结论　所筛选出的FXMcAb为高纯度FX的提取和缺乏FX血浆的制备以及FX抗原的ELISA检测方法的建立 ,提供了试剂准备 .更多还原

【Abstract】 Objective To prepare high purity coagulation facton X(FX) and FX deficient plasma and to develop ELISA for the human FX antigen.Methods Partially purified preparations of homan FX were used to immunize Balb/C mice by twice basic immunization and one booster immnunization,the speleen cells of these animals were fused with mouse myeloma cell line SP2/0-Ag14 cells. The hybridoma strains were screened by indirect ELISA method. The spectificity analysis was carried by Western-blotting test,Cross-reaction test, Addition test, Divalent metal ions dependent test and so on.Results After 3 to 4 limiting recloning. Ten mouse hybridomas with stable secreting ability were obtained. All antibodies are the IgG1 subclass. The titers of all McAbs from ascites were over 106. The 10 McAb conformation-specific monoclonal antibodies, and the binding to FX is inhibited in the presence of Zn 2+ .Conclusion The screened monoclonal antibodies could make the ragent preparation for FX deficient plasma preparation and high purify FX preparation by immuno-affinity chromatography and ELISA for the human coagulation FX antigen.更多还原