人纤溶酶活性动态显色检测方法的建立及验证  

Development and verification of dynamic chromogenic method for determination of human plasmin activity

作　　者：岳胜兰 周志军[1] 彭焱[1] 林连珍[1] 李陶敬[1] 邢延涛[1] 汪菲菲[1] 李策生[1] 胡勇[1] YUE Sheng-lan ZHOU Zhi-jun PENG Yan LIN Lian-zhen LI Tao-jing XING Yan-tao WANG Fei-fei LI Ce-sheng HU Yong(Wuhan Institute of Biological Products Co. , Ltd. , Wuhan 430207, Hubei Province, Chin)

机构地区：[1]武汉生物制品研究所有限责任公司血液制剂研究室,湖北武汉430207

出　　处：《中国生物制品学杂志》2017年第1期74-78,共5页Chinese Journal of Biologicals

基　　金：国家863计划(2012AA021904);2013年湖北省重大科技创新计划(2013ACC001);武汉国际科技合作计划(2015030809020364)

摘　　要：目的建立人纤溶酶(plasmin,Plm)活性的动态显色检测方法,并进行验证。方法用微量滴定板作为载体,将不同活性浓度的Plm(1.0、0.5、0.25、0.125、0.062 5、0.031 25 IU/ml)分别与不同浓度的发色底物S-2251(2.0、1.0、0.66、0.33 mg/ml)混匀,连续监测10 min,测定反应体系吸光值变化率(ΔA/min),确定方法的反应参数。同时对方法的选择性、线性范围、定量下限、准确度、精密性、特异性及稳定性进行验证。采用建立的方法检测Plm纯化样品。结果确定定量上限为0.6 IU/ml,底物浓度为0.66 mg/ml;监测5 min时校正标样及质控样品回收率为92.0%~111.8%,定量下限样品回收率为96.5%~118.0%。注射用水、稀释液、尿激酶(Urokinase,UK)、6-氨基己酸(6-Aminocaproic acid,EACA)、人纤溶酶原(plasminogen,Plg)等组分的响应低于定量下限响应的8.22%,并低于内标响应的0.86%,表明该方法具有良好的选择性;线性范围为0.05~0.6 IU/ml,校正标样回收率在96.8%~111.2%之间,R2≥0.99;定量下限为0.05 IU/ml,定量下限处准确度在115.2%~116.2%之间,CV≤5.0%;高、中、低浓度质控样品检测结果准确度在102.3%~111.8%之间;批内CV值≤7.7%,批间CV值≤5.2%;0.5μg/ml UK对Plm活性检测无影响,EACA浓度在0.05~0.2 mol/L之间及Plg浓度低于0.3 mg/ml时对Plm活性检测结果无影响;Plm质控样品复溶后在室温及2~8℃放置1 h不影响检测结果。Plm纯化样品活性回收率为91.84%。结论本方法具有良好的准确度、精密度、特异性及稳定性,可用于工艺样品中Plm活性的检测。Objective To develop and verify a dynamic chromogenic method for determination of human plasmin（Plm）activity. Methods Using microtiter plate as a carrier, the chromogenic substrate S-2251 at various concentrations（2. 0,1. 0, 0. 66 and 0. 33 mg/ml）were incubated with plasmin at various concentrations（1. 0, 0. 5, 0. 25, 0. 125, 0. 062 5 and0. 031 25 IU/ml）, monitored for 10 min constantly, and determined for ΔA/min, based on which the assay parameters were determined. The dynamic chromogenic assay was verified for selectivity, linear range, lower limit of quantitation（LLOQ）, accuracy, precision, specificity and stability, and used for determination of purified Plm. Results The upper quantitative limit and substrate concentration of the developed method were 0. 6 IU/ml and 0. 66 mg/ml respectively.The recovery rates of calibration standards and quality controls after being monitored for 5 min were 92. 0 %~ 111. 8%,while that of samples at a concentration of LLOQ was 96. 5% ~ 118. 0%. The response values of water for injection（WFI）, diluent, urokinase（UK）, EACA and plasminogen（Plg） were lower than 8. 22% of that of samples at a concentration of LLOQ and 0. 86% of that of internal standard, indicating high selectivity of the method. The linear range of the method was 0. 05 ~ 0. 6 IU/ml, while the recovery of calibration standards was 96. 8% ~ 111. 2% with a R2 value of not less than 0. 99. The LLOQ was 0. 05 IU/ml, while the accuracy of the method for samples at a concentration of LLOQ was 115. 2% ~ 116. 2%, with a CV of less than 5. 0%. The accuracies of the method for quality control samples at high, moderate and low concentrations were 102. 3% ~ 111. 8%, of which the CVs in intra-and inter-assays were not more than 7. 7% and not more than 5. 2% respectively. The UK at a concentration of 0. 5 μg/ml, EACA at a concentration of 0. 05 ~ 0. 2 mol/L and Plg at a concentration of less than 0. 3 mg/ml showed no influence on the determination of Plm activity. The reconstituted quality control s

关 键 词：人纤溶酶 动态显色法 酶活性 

分 类 号：R392-33[医药卫生—免疫学]