凝血因子IX的体外检测方法研究

Study on in vitro assay methods for human coagulation factor IX

凝血因子ＩＸ的检测是血友病Ｂ基因治疗研究中的重要工作，在实验室原有基础上，发展和完善了凝血因子ＩＸ在蛋白质水平上的检测系统，为基因治疗血友病Ｂ提供了更为直观可靠的依据．首先，建立了以鼠抗人ＦＩＸ单克隆抗体Ａ－７为一抗的检测活性ＦＩＸ蛋白量的ＥＬＩＳＡ法，为检测活性ＦＩＸ提供了快速简便的方法．其次，实现了用Ｗｅｓｔｅｒｎｂｌｏｔ法检测转染细胞培养液上清中ＦＩＸ，确证了体外培养的转有人ＦＩＸｃＤＮＡ的细胞表达有人ＦＩＸ蛋白．最后，用免疫组织化学方法，原位检测了转有人ＦＩＸｃＮＡ的细胞表达的ＦＩＸ蛋白，展现了在细胞水平上ＦＩＸ表达的直观图象．

he assay of human factor IX is important in the study of hemophilia B gene therapy. Previous methods developed in this lab were improved on the protein level,and a much more reliable and directly perceivable one was found. Firstly,we had established an ELISA method testing active factor IX using mouse antihuman FIX monoclonal antibody A-7 as first antibody,which resulted a quicker and easier active FIX testing. Secondly,we had carried out hFIX assay in the cell culture medium by developing a Western blot method. Fingally,we had established an immunohistochemistry method to test the FIX expressing cells transfected with an adenovirus vector carrying FIX cDNA. The present method provides a directly perceivalbe image of FIX expression on the cell level