人血浆凝血因子Ⅶ单克隆抗体的制备与应用初探

Preparation and purification of monoclonal antibody against human coagulation factor Ⅶ

目的制备能稳定分泌人凝血因子Ⅶ(FⅦ)单克隆抗体的杂交瘤细胞株,纯化人FⅦ单克隆抗体并初步应用。方法 1)以纯化的人血浆FⅦ为抗原免疫BALB/c小鼠,用杂交瘤技术筛选出能分泌人FⅦ单克隆抗体的细胞株,用体内诱生法制备小鼠腹水,经硫酸铵沉淀、DEAE-affi-gel-blue凝胶层析纯化单克隆抗体;鉴定抗体的纯度和亚类、抗体与FⅡ、FⅨ、FⅩ的交叉反应性、二价金属离子对抗原抗体结合的影响等特性。2)将人FⅦ单克隆抗体连接到GoldMag-@磁微粒表面,检测连接效率;将磁微粒连接的抗体与FVⅡ反应,并用含有二价金属离子的缓冲液进行洗脱,探索用磁微粒-单克隆抗体免疫亲和吸附法纯化FⅦ的方法和可行性。结果 1)成功制备出22株分泌人FⅦ单克隆抗体的杂交瘤细胞株,命名为HFⅦ001～022;所选取的HFⅦ002、0040、050、130、140、21等6株细胞制备的纯化的抗体,其重链的亚类均为IgG1,除与FⅦ反应外,均不与FⅡ、FⅨ、FⅩ发生交叉反应,其中HFⅦ005可以抑制血浆FⅦ的凝血活性,HFⅦ0020、040、05与FⅦ的结合明显受Zn2+的抑制。2)上述6株细胞抗体与磁微粒的连接率为24.0%～94.7%,HFⅦ0020、050、13等3株细胞抗体连接磁微粒后可以结合FⅦ,对其中1株磁微粒连接抗体(HFⅦ005)进行试验显示:其结合的FⅦ在有Zn2+存在时能解离下来。结论制备出能分泌人血浆FⅦ单克隆抗体的杂交瘤细胞株并纯化出单克隆抗体;应用磁微粒-单克隆抗体免疫亲和吸附法,可以分离纯化人FⅦ。

Objective To prepare hybridoma cell lines which can secrete monoclonal antibody against human coagulation factor Ⅶ（FⅦ） and to purify the monoclonal antibodys against FⅦ.Methods（1）BALB/c mice were immunized with purified human FⅦ.Hybridoma cell lines which were able to secrete monoclonal antibody against FⅦ were prepared by hybridoma technique.Hybridoma cells were injected to the abdominal cavity of BALB/c mice for induction of ascites.Monoclonal antibodies were purified from ascites fluid with ammonium sulfate fractionation and DEAE-affi-gel-blue chromatography.Then the purity and subclasses of the monoclonal antibody were identified.The characteristics of these antibodies were studied,including the cross reaction between the monoclonal antibody and coagulation factor Ⅱ（FⅡ）,factor Ⅸ（FⅨ）,and factor Ⅹ（FⅩ）,and the effect of divalent metal ion on the combination between monoclonal antibody and FⅦ.（2）The purified monoclonal antibodies were coupled to GoldMag-@ magnetic particulate and the efficiency of couple reaction was detected.The antibodies coupled to magnetic particulate reacted with FⅦ,and then the bound FⅦ was eluted from the surface of magnetic particulate with the buffer containing bivalent metal ion.The feasibility of purifying FⅦ with the monoclonal antibody was evaluated.Results（1） 22 hybridoma cell lines were obtained and named as HFⅦ-001 to HFⅦ-022.Six monoclonal antibodies were purified from ascites which were induced by six selected hybridoma cell lines（HFⅦ-002,004,005,013,014,021）.And all the six antibodies had no cross reactions with FⅡ,FⅨ,and FⅩ.An antibody（HFⅦ-005） could inhibit the clotting activity of FⅦ in plasma.Zn2＋ could remarkably restrain the combination of FⅦ with three antibodies against FⅦ（HFⅦ-002,004,005）.（2） The six antibodies were efficiently coupled with GoldMag-@ magnetic particulate.The binding rates were 24.0%~94.7%.Three antibodies（HFⅦ-002,004,013） could combine with FⅦ after coupling with magnetic particulate,and one of the antibodies（HFⅦ-005） could be eluted by the buffer containing Zn2＋ to obtain purified FⅦ.Conclusion Twenty two hybridoma cell lines which can secrete monoclonal antibodies against FⅦ are prepared and purified.The purified antibodies coupled with magnetic particulate could be used to purify FⅦ.