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抗组织因子途径抑制物单克隆抗体的制备及鉴定
引用本文:杨寅柯 何晓凡. 抗组织因子途径抑制物单克隆抗体的制备及鉴定[J]. 中华血液学杂志, 1997, 18(3): 118-122
作者姓名:杨寅柯 何晓凡
作者单位:湖南医科大学生理教研室
摘    要:目的:制备抗组织因子途径抑制物(TFPI)单克隆抗体。方法:用微量TFPI脾内包埋免疫BALB/c小鼠,取脾细胞与SP2/O细胞融合,建立了2株稳定分泌抗TFPI单克隆抗体(McAb)的杂交瘤细胞株,分别命名为4F4和4F8。对其中的一株4F8进行研究,其杂交瘤细胞染色体众数为93,分泌的抗体为IgG1。以简易正辛酸法从腹水纯化McAb4F8,SDS-PAGE检测其纯度,Westernbloting分析显示其可特异性地识别分子量为34800的抗原分子。对纯化的抗体以改良过碘酸钠氧化法用辣根过氧化物酶标记后,采用双夹心ELISA法检测正常人血浆TFPI含量。结果:稀释的凝血酶原时间(PT)测定,McAb4F8可缩短其凝固时间,且呈量效关系,McAb4F8还可使乏因子Ⅸ血浆稀释的PT明显缩短。正常人血浆TFPI含量为103.2±11.5μg/L,结果与美国Diagnostica公司的TFPIELISA试剂盒所测结果(98.4±10.3μg/L)相近(r=0.92)。结论:McAb4F8有潜在的药用价值,并可望为我国TFPI的研究提供精确的检测手段。

关 键 词:抗体.单克隆  杂交瘤  组织因子途径抑制物

Preparation and characterization of a McAb against tissue factor pathway inhibitor
Yang Yinke,He Xiaofan,Li Juncheng,et al.. Preparation and characterization of a McAb against tissue factor pathway inhibitor[J]. Chinese Journal of Hematology, 1997, 18(3): 118-122
Authors:Yang Yinke  He Xiaofan  Li Juncheng  et al.
Affiliation:Department of Physiology, Hunan Medical University, Changsha 410078.
Abstract:OBJECTIVE: To prepare monoclonal antibody (McAb) against tissue factor pathway inhibitor (TFPI). METHODS: Twice intrasplenic immunization of BALB/c mice with TFPI-binded nitrocellulose paper followed by an intraperitoneal boost with TFPI raised two hybridoma cell lines, 4F8 and 4F4, by hybridoma technique, 4F8 was studied in detail. The mode value of its chromosome is 93. The Ig class of McAb 4F8 was IgG1. The McAb was purified from ascites by a simple procedure of caprylic acid pre cipitation. The purified McAb was conjugated with horseradish peroxidase (HRP) by a modified periodate oxidation procedure. RESULTS: Immunoblotting indicated that the McAb recognized a single band of TFPI with a molecular weight of 34800. The McAb was also proved to shorten markedly the dilute thromboplastin coagulation time of factor IX-deficient and normal plasma. The results of Sandwich ELISA using the HRP labeled McAb showed that the mean TFPI in normal human plasma is 103.2 +/- 11.5 microg/L, being similar to that measured with TFPI ELISA kit from America Diagnostica Inc. (98.4 +/- 10.3 microg/L) (r=0.92). CONCLUSION: McAb 4F8 may be a useful reagent for TFPI research.
Keywords:Antibody  monoclonal Hybridoma Tissue factor pathway inhibitor  
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