人血浆中纤维蛋白溶酶原纯化工艺样品蛋白含量改良BCA检测方法的建立及验证  

Development and verification of a modified BCA method for determination of plasminogen content in human plasma

作　　者：胡勇 纪德铭 詹骞 陈克金 彭焱 周雁翔 岳胜兰 李娟 周志军 HU Yong;JI De-ming;ZHAN Qian;CHEN Ke-jin;PENG Yan;ZHOU Yan-xiang;YUE Sheng-lan;LI Juan;ZHOU Zhi-jun(Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]国药集团武汉血液制品有限公司,湖北武汉430207

出　　处：《中国生物制品学杂志》2021年第5期595-601,共7页Chinese Journal of Biologicals

摘　　要：目的建立检测人血浆中纤维蛋白溶酶原(plasminogen,Pg)改良PIerceTMBCA蛋白试剂盒方法(简称改良BCA法),并进行验证及初步应用。方法结合PierceTMBCA试剂盒的试管法与微孔板法检测人血浆中Pg;比较标准曲线拟合方式确定改良试剂盒的线性范围;通过牛血清白蛋白(bovine serum albumin,BSA)国家标准品和PIerceTM BCA试剂盒附带的BSA标准品互相标定确定内控标准品;通过平行性试验分析BSA作为标准品检测Pg蛋白含量的可行性。验证方法的准确性、精密性及选择性。应用改良的BCA法检测Pg纯化过程的蛋白含量,并与凯氏定氮法和双缩脲法进行比较,同时检测201903003批Pg工艺纯化过程样品蛋白含量的相对纯度及比活性变化。结果改良BCA法选择二次函数拟合,PIerceTMBCA试验盒线性范围为62.5~2000μg/mL,试剂盒附带的BSA标准品(2000μg/mL)用BSA国家标准品标定结果为2090μg/mL,可作为内控标准品使用;Pg所在工艺组分浓度在250~2000μg/mL时,内控BSA曲线与Pg曲线的平行性最好。内控标准品的高(1500μg/mL)、中(750μg/mL)、低(150μg/mL)、检测下限(62.5μg/mL)及BSA国家标准品(2000μg/mL)的回收率在88.61%~103.78%之间,批内及批间CV均<10%;Pg工艺缓冲液中150 mmoL/L辛酸钠、25 g/L蔗糖、5 g/L甘氨酸、3%盐酸精氨酸对检测无影响,1%溶剂/去污剂(S/D)稀释至少40倍对检测无干扰。改良BCA法检测Pg纯化工艺的原液、半成品结果与双缩脲法和凯氏定氮法的符合性良好。201903003批工艺样品从原料血浆经过3步纯化后获得Pg半成品,其比活性从0.02 U/mg升至8.42 U/mg,相对纯度从0.19%升至81.94%。结论成功建立了改良的BCA蛋白定量检测方法,该方法线性范围广,平行性、准确性、精密性及选择性良好,既可用于Pg纯化工艺中间品的质量监控,也可用于原液、半成品的蛋白含量检测。Objective To develop,verify and preliminarily apply a modified PierceTMBCA protein quantitative analysis kit for determination of plasminogen(Pg) content in human plasma. Methods The Pg content in human plasma was determined by the tube assay combined with microplate assay in PierceTMBCA kit. The method was validated for linear range by comparison of fitting methods of standard curves. The internal control standard was determined by the cross calibration of national standard for bovine serum albumin(BSA)and the standard BSA in PierceTMBCA kit. The feasibility of BSA as a standard in determination of Pg content was analyzed by parallelism test. The developed method was validated for accuracy,precision and selectivity,by which the Pg content during purification was determined,and the result was compared with those by Kjeldahl method and biuret method. The Pg samples taken from the whole process were determined for relative purity and specific activity by the developed BCA method. Results Quadratic function fitting showed that the linear range of the modified BCA method was 62. 5 ~ 2 000 μg/mL. The standard bovine serum albumin(BSA)included in the kit was calibrated as 2. 090 μg/mL with national standard for BSA,which might be used as an internal control st andard. When the concentration of component containing Pg was 250 ~ 2 000 μg/mL,the curve of internal control BSA showed good parallelism to that of Pg. The recovery rates of international control standard at concen-trations of 1 500,750,150 and 62. 5 as well as national at a concentration of 2 000 μg/m L were 88. 61% ~ 103. 78%,and the both the CVs in intra-and inter-assays were less than 10%. The 150 mmol/L sodium octanoate,25 g/L sucrose,5 g/L glycine and 3% arginine hydrochloride in the process buffer showed no effect on,while the 1% S/D diluted at least 40 folds showed no interference to,the determination result. The determination results of bulk and final bulk of by the modified BCA method were consistent with those by biuret method and Kjeldahl method.

关 键 词：改良BCA法 人纤维蛋白溶酶原 蛋白含量 平行性分析 过程监控 

分 类 号：R973.2[医药卫生—药品]