人凝血酶原复合物中人凝血因子Ⅺ残留量检测方法验证  

Validation of the detection method for residual human coagulation factorⅪin human prothrombin complex

作　　者：刘勇 余雨蓉 杨龙 李泽秀 张尧 邓靖 李丹 陈云华 赵学梅 LIU Yong;YU Yurong;YANG Long;LI Zexiu;ZHANG Yao;DENG Jing;LI Dan;CHEN Yunhua;ZHAO Xuemei(GuiZhou Tai-Bang Biological Products Co.Ltd.,Guiyang 550025,China)

机构地区：[1]贵州泰邦生物制品有限公司,贵州贵阳550025

出　　处：《中国输血杂志》2023年第9期818-822,共5页Chinese Journal of Blood Transfusion

摘　　要：目的 建立酶联免疫吸附法(ELISA)测定人凝血酶原复合物中人凝血因子Ⅺ残留量的方法,并进行方法验证。方法 人凝血因子Ⅺ将与包被在微滴定板上的捕获抗体结合并发生反应。经过适当的洗涤步骤后,生物素一抗结合到捕获的蛋白质上。过多的一抗被洗掉,结合的抗体与辣根过氧化物酶标记的链霉亲和素反应,加入TMB底物显色,置于酶标仪450 nm波长处检测。对该检测方法的稀释可靠性、准确度、专属性、重复性、中间精密度、线性、范围、耐用性进行验证。结果 该方法准确度、专属性良好,平均回收率为109.2%, RSD值为6.93%;重复性RSD为6.78%,中间精密度RSD为6.75%,精密度良好;线性相关系数r=0.999 9,线性范围内准确度、精确度良好;耐用性验证了孵育时间和试剂盒开启有效期,结果显示孵育时间变化RSD为6.62%,说明该检测方法孵育时间控制在28~32 min,对结果无显著影响;试剂盒开启后按条件保存7 d,前后检测结果RSD为3.84%,说明试剂盒开启后按条件保存7 d对FⅪ检测结果无影响;上述两者结果表明该方法耐用性良好。稀释可靠性结果显示该检测方法检测PCC中FⅪ残留量时存在“钩状”效应,可以通过稀释100~200倍解决。结论 该方法能够满足实验室人凝血酶原复合物中人凝血因子Ⅺ残留量的测定。Objective To establish an enzyme⁃linked immunosorbent assay(ELISA)method for the determination of re⁃sidual human coagulation factorⅪin human prothrombin complex and validate the method.Methods Human factorⅪwas reacted with the capture antibody coated on the microtiter plate.After appropriate washing steps,biotinylated primary anti⁃body was bound to the captured protein.Excess primary antibody was washed away and bound antibody was reacted with horseradish peroxidase conjugated streptavidin.TMB substrate was used for color development at 450 nm.The dilution relia⁃bility,accuracy,specificity,repeatability,intermediate precision,linearity,range and durability were verified.Results The verification results showed that the accuracy and specificity of this method met the experimental requirements,with an average recovery rate of 109.2%and RSD of 6.93%.The repeatability RSD was 6.78%,and the intermediate precision RSD was 6.75%,indicating good precision.The linear regression correlation coefficient of standard curve was 0.9999,showing good accuracy and precision within the linear range.The durability was verified by the incubation time and the validity period of reagent kit opening.The results showed that the RSD of the incubation time change was 6.62%,indicating that the incuba⁃tion time of this detection method was controlled between 28 to 32 minutes,and there was no significant impact on the re⁃sults.The RSD of the detection results before and after the reagent kit was opened and stored under conditions for 7 days was 3.84%,indicating that the preservation of the reagent kit according to the conditions for 7 days after opening has no effect on the FⅪdetection results.Both indicated that the method had good durability.The dilution reliability results showed that there was a"hook"effect in the detection of FⅪresidue in human prothrombin complex,which could be solved by diluting 100 to 200 times.Conclusion This method can be used for the determination of FⅪresidues of human prothrombin complex in

关 键 词：人凝血酶原复合物 人凝血因子Ⅺ 酶联免疫吸附法 

分 类 号：R554.9[医药卫生—血液循环系统疾病]