发色底物检测法用于人纤维蛋白溶酶原纯化工艺的可行性分析

Feasibility of application of chromogenic substrate method to purification process of human plasminogen

目的分析发光底物检测法用于人纤维蛋白溶酶原(plasminogen,Pg)纯化工艺的可行性。方法采用纤溶酶原测定试剂盒对ACL7000血凝仪运行发色底物法的性能进行确认;并对该方法的线性范围、准确度、精密度、选择性进行验证;应用Softmax及Statistic软件分析定标血浆及Pg纯化样品检测结果的平行性;采用验证的方法检测Pg全层析工艺样品的效价及目标样品存贮稳定性。结果ACL7000血凝仪运行发色底物法的性能稳定。定标血浆标准曲线线性范围为0.128~1.28 U/mL,稀释样品回收率在92%~103%之间,R2≥0.99;定标血浆的高、中、低值、检测下限及正常质控血浆(control plasma normal,CPN)效价回收率批内及批间分别在91%~108%和94%~107%之间,批内及批间CV分别在0.5%~1.9%和0.3%~1.4%之间;Pg纯化各段层析缓冲液所含特殊组分除1%Tween80+0.3%磷酸三丁酯外对Pg效价检测无影响。纯化样品Pg效价检测曲线与定标血浆曲线平行。全层析工艺Pg半成品的比活性达8 U/mg以上,效价总回收率达50%以上;Pg亲和层析洗脱液在-30℃保存42 d、18~25℃及2~8℃保存7 d效价稳定,Pg半成品在-30℃保存68 d、18~25℃及2~8℃保存38 h效价稳定。结论纤溶酶原测定试剂盒(发色底物法)具有较好的线性范围、准确度、精密度和选择性,ACL7000血凝仪运行该方法可用于Pg全层析工艺样品的效价检测,本实验为Pg纯化工艺研究奠定了基础。

Objective To analyze the feasibility of chromogenic substrate method for determination of human plasminogen(Pg)potency to purification process of Pg.Methods The performance of ACL7000 blood coagulation instrument running the chromogenic substrate method was confirmed by HemosiL Pg activity detection kit,and the linear range,accuracy,precision and selectivity of the method were validated.The parallel analysis between purified samples and calibration plasma was performed by using Softmax and Statistic software.The confirmed method was used for the detection of Pg potency in full chromatographic process samples and the determination of stability of target samples in storage.Results The performance of ACL7000 blood coagulation instrument was stable in chromogenic substrate method.The linear range of standard curve was 0.128~1.28 IU/mL,while the recovery rates of diluted samples were 92%~103%(R2≥0.99).The accuracies of standards at high,moderate and low concentrations and the minimum detection limit sample and CPN in intra-and intera-ssays were 91%~108%and 94%~107%,while the CVs of potency were 0.5%~1.9%and 0.3%~1.4%,respectively.Except 1%Tween 80+0.3 Tributyl phosphate,the specific components contained in buffer showed no effect on the determination of Pg potency.The detective curves of Pg potency in purified samples and CPN were parallel to the standard curves of calibration plasma.The specific activity of final bulk of Pg in the full chromatographic process reached more than 8 U/mg,while the total recovery rate of potency reached more than 50%.The potency of eluent of Pg from affinity chromatography was stable after storage at-30℃for 42 d and at 18~25 and 2~8℃for 7 d.However,the potency of final bulk of Pg was stable after storage at-30℃for 68 d and at 18~25 and 2~8℃for 38 h.Conclusion The Pg detection kit(chromogenic substrate method)showed good linear range,accuracy,precision and selectivity.The method run by ACL7000 blood coagulation instrument may be used for determination of potency of Pg in full chromatographic process samples.The study laid a foundation of research on purification process of Pg.