抗组织因子途径抑制物单克隆抗体的制备及鉴定

Preparation and characterization of a McAb against tissue factor pathway inhibitor

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。

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 IgG 1. The McAb was purified from ascites by a simple procedure of caprylic acid precipitation.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±11.5μg/L, being similar to that measured with TFPI ELISA kit from America Diagnostica Inc.(98.4±10.3μg/L)(r=0 92). Conclusion:McAb 4F8 may be a useful reagent for TFPI research.