摘要
经凝胶过滤和亲和层析分离得到的人抗血友病A因子——VIII因子抗原VIII:CAg,在用一期法和火箭免疫电泳证实其纯度后,免疫Balb/c小鼠和包被免疫测定板。采用常规鼠杂交瘤技术经间接ELISA法筛选、克隆化得到四个稳定分泌抗VIII:C的单克隆抗体的细胞株,即DC8,DE4,DE7和DE8,腹水效价为1:1×10~4～1:1×10~5,一期法抑制反应表明,单克隆抗体对VIII:C活性有显著的抑制作用,但其抑制是不完全的,对抗体进行了纯化及分析,并将单抗用于血友病A多态性的分析。
The VIII:CAg (containing the coagulant activity) and VWF (Von WillebrandFactor) were seperated from human FVIII preparations (partly-purified fromhuman plasma) by gel filtration and affinity chromatography. One-stage assay forVIII:C and the rocket immunoelectrophoresis were used to check its purity. Balb/cmice were immunized with the purified VIII:CAg, and the mouse spleen cells weresuccessfully fused with sp2/o myeloma cells induced by PEG. After selection withthe indirect ELISA, four cell lines, namely DC8, DE4, DE7, and DE8, were ob-tained and cloned that stably secreted antiVIII:C antibodies. Except the DE4 cellswhich induced solid tumor, the other three hybridoma cells developed ascitic fluidsin mice and the titres of antibodies in ascites were found to be 1:1×10~4~1:1×10~6,By one-stage inhibition assay (biological activity assay for McAbs), it was shownthat the McAbs could strongly, but not completely, inhibit the VIII:C activity inhuman plasma. The production of McAbs in vitro, ie., in serum-free culture, itspurification identification, of the subclasses, McAbs′homogenerasity and molecularweight, were also carried out. Finally, the first-step application of the preparedMcAbs in clinical diagnosis for hemophilia A polymorphism was discussed.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
1991年第1期62-69,共8页
Journal of Fudan University：Natural Science
基金
国家863高技术项目基金资助课题
关键词
血友病
血凝抗体
单克隆抗体
hemophilia, coagulation antibody, immunoassay
human coagulant FVIII:C, one-stage assay.
