酶标仪法测定离心泵泵头表面肝素涂层生物活性的研究  

Study on the bioactivity of heparin coating on the surface of centrifugal pump head by an enzyme-labeled method

作　　者：徐苏华[1] 刘婧群 许朝生 黄敏菊[1] Xu Suhua;Liu Jingqun;Xu Chaosheng;Huang Minju(Guangdong Medical Devices Quality Surveillance and Test Institute,NMPA Key Laboratory of Extracorporeal Circulation Devices,Guangdong Guangzhou 510663,China)

机构地区：[1]广东省医疗器械质量监督检验所国家药品监督管理局体外循环器械重点实验室,广州510663

出　　处：《中国体外循环杂志》2024年第5期415-419,共5页Chinese Journal of Extracorporeal Circulation

基　　金：国家重点研发计划(2023YFC2410304);广东省药品监督管理局科技创新项目(2024ZDZ05)。

摘　　要：目的 建立酶标仪法测定离心泵泵头表面肝素的生物活性。方法 由抗凝血酶(AT)饱和的肝素化表面失活的凝血酶量来量化肝素生物活性:肝素表面与过量的血浆蛋白AT结合,形成AT饱和的肝素化表面(过量的AT通过清洗程序去除),上述肝素化表面又与过量凝血酶Ⅱa结合,残留的凝血酶Ⅱa与显色底物反应,凝血酶肽键H-D-Phe-Pip-Arg-pNa被裂解,释放出生色团,其可以在405 nm下进行测试并定量。结果 应用酶标仪上波长405 nm时的动力学吸光度评估生物活性,生物活性测定表明:对照品(无涂层的离心泵泵头)的结果为-0.01 IU/cm^(2)(≤0.05 IU/cm^(2)),所有3种供试品的结果为0.29 IU/cm^(2)、0.40 IU/cm^(2)和0.32 IU/cm^(2)(均≥0.1 IU/cm^(2)),符合生物活性试验的有效性标准,对照品和供试品的生物活性均被视为通过。结论 通过酶标仪定量测试凝血酶肽键裂解释放生色团的含量,可得到与肝素涂层反应的Ⅱa的量,进一步反推AT的量,最终计算出肝素涂层的生物活性。该方法操作简便,一次可以测定多份样本,检测速度快;具有需要的样本量少、重复性好的特点,适用于对离心泵泵头表面肝素涂层的生物活性定量检测。Objective To establish an enzyme-linked immunosorbent assay(ELISA)method for assessing the biological activity of heparin on the surface of centrifugal pump heads.Methods The method quantifies the biological activity of heparin by measuring the amount of thrombin inactivated on the heparinized surface saturated with antithrombin(AT).The surface of heparin binds to an excess of plasma protein AT to form an antithrombin-saturated heparinized surface(excess AT is removed through a cleaning program),which in turn binds to an excess of thrombin(Ⅱa).The remaining thrombin(Ⅱa)reacts with the chromogenic substrate,and the peptide bond H-D-Phe Pip-Arg-pNa of thrombin is cleaved,releasing chromophores that can be tested and quantified at 405 nm.Results The kinetic absorbance at a wavelength of 405 nm on an enzyme-linked immunosorbent assay(ELISA)could be used to evaluate biological activity.Bioactivity measurements showed that the result of the control sample(uncoated centrifugal pump head)was-0.01 IU/cm^(2)(≤0.05 IU/cm^(2)),and the results of all three test samples were 0.29 IU/cm^(2),0.40 IU/cm^(2),and 0.32 IU/cm^(2)(all≥0.1 IU/cm^(2)),which met the validity criteria of the biological activity test.The biological activity of both the control and test samples was considered to have passed in terms of their biological activity levels.Conclusion By quantitatively testing the content of chromophores released by thrombin peptide bond cleavage using an enzyme-linked immunosorbent assay(ELISA)reader,the amount of IIa reacting with heparin coating can be obtained,and the amount of AT can be further inferred to ultimately calculate the biological activity of heparin coating.This method is easy to operate,allows for simultaneous measurementof multiple samples,and has fast detection speed.The characteristics of requiring small sample size and good repeatability make it suitable for quantitative biological activity detection of heparin coatings on the surface of centrifugal pump heads.

关 键 词：酶标仪法 离心泵泵头 肝素生物活性 抗凝血酶 凝血酶 体外膜氧合 

分 类 号：TH77[机械工程—仪器科学与技术]