缺凝血因子Ⅶ血浆的研制及初步性能评价

Production and primary function evaluation of coagulation factor Ⅶ deficient plasma

目的制备缺凝血因子Ⅶ血浆(FⅦDP)并初步评价该FⅦDP的性能。方法利用本实验室建立的抗人凝血因子Ⅶ单克隆抗体(FⅦMcAb)杂交瘤细胞株,采用小鼠体内诱生腹水法和亲和层析法制备高亲和力的FⅦMcAb,并将其偶联至CNBr-Activated Sepharose 4B上,制成FⅦMcAb免疫亲和层析柱,制备FⅦDP;采用凝血一期法检测自制FⅦDP中各凝血因子促凝活性并考察自制FⅦDP的精密度,对比自制FⅦDP和同类进口FⅦDP制品的测定线性范围、比较二者检测不同FⅦ水平样品的效果。结果所制备的FⅦMcAb亚类均被鉴定为IgG1型,利用该FⅦMcAb制备的FⅦDP中FⅦ促凝活性(FⅦ∶C)<1%,而FⅡ∶C、FⅤ∶C、FⅧ∶C、FⅨ∶C及FⅩ∶C都>70%,均满足正常凝血所需水平;该FⅦDP线性范围在1.625%—200%,3批次自制FⅦDP批内精密度(Sr)分别为3.30、3.36和3.30,批内变异系数分别为4.12%,4.11%,3.98%;批间精密度(Srr)4.20,批间变异系数4.99%,标准曲线的相关系数(r)分别为-0.998、-0.996和-0.996与同类进口制品(r=-0.998)基本一致;经相关分析,该FⅦDP对于不同FⅦ水平的样品的检测效果与同类进口产品一致,r分别为0.97、0.96和0.97。结论初步评价显示FⅦMcAb免疫亲和层析柱研制出FⅦDP性能良好,可作为FⅦ检测试剂使用。

Objectives To produce coagulation factor Ⅶ deficient plasma （FⅦDP） and evaluate the function of the plasma. Methods A high affinity monoclonal antibody （McAb） of factor Ⅶ was obtained from ascitic fluid of BALB/c mice in which the antibody-producing cells had been grown as ascitis tumor. Then, FⅦMcAb was purified from ascitic fluid using HiTrap rProtein A HP. The McAb was coupled with CNBr-activated Sepharose 4B to form an affinity chromatographic column. The FⅦDP was obtained when normal fresh plasma passed through the column. Coagulation factors concentrations were measured by the methods of one-stage coagulation assays. To evaluate the precision of the immunodepleted plasma substrate,the linear ranges of the immunodepleted plasma substrate were investigated, comparing with imported commercial products. Results The isotype of the FⅦMcAb prepared by HiTrap rProtein A affinity chromatography was identified as IgG1. The FⅦDP were produced by using affinity chromatographic column coupled with the artificial FⅦMcAb. The FⅦ in this biodepleted FⅦDP gained was removed completely （FⅦ∶C1%） and the amount or activity of other coagulation factors remained unchanged （FⅡ∶C,FⅤ∶C,FⅧ∶C,FⅨ∶C and FⅩ∶C〉70%）.The precisions （Sr） and coefficients of variation （CV） of three batches of production within batches were 3.30, 3.36, 3.30 and 4.12%, 4.11%, 3.98%, respectively, while the between-run precision （Srr）and coefficient of variation （CV） are 4.20 and 4.99%, respectively. The linear range of three batches of production and the measured results of a series of samples were evaluated by making a contrast with congeneric commercially available FⅦDP. The preliminary evaluation indicates that this biodepleted substrate was of high precision and with broad linear range. Moreover, it gave an excellent standard curve （related coefficients are -0.998, -0.996, -0.996, respectively） over a wide range of normal plasma dilutions （1.625%—100%） and a good correlation of FⅦ：C concentrations with those obtained using a commercial substrate （r=-0.998） for a series of samples （r=0.97,r=0.96,r=0.97）. Conclusion Preliminary evaluation shows that the plasma could be used as a substrate in a one stage coagulation assay and performed as well as commercially available FⅦDP.