重组假丝酵母尿酸氧化酶等电点毛细管等电聚焦检测方法的建立及验证

Development and verification of capillary isoelectric focusing method for determination of isoelectric point of recombinant Candida uricase

目的建立检测重组假丝酵母尿酸氧化酶等电点(isoelectric point,pI)的毛细管等点聚焦(capillary isoelectrie focusing,cIEF)方法,并进行验证。方法通过优化cIEF过程中稳定剂比例、聚焦时间等参数,建立cIEF方法,并对方法的专属性、精密度、准确性、耐用性进行验证。结果确定稳定剂精氨酸(arginine,ARG)︰亚氨基二乙酸(iminodiacetic acid,IDA)比例60μL︰6μL,聚焦时间15 min为最适条件。样品基质在重组假丝酵母尿酸氧化酶主峰位置无干扰峰;重复性、中间精密度、批间精密度验证相对标准偏差(relative standard deviation,RSD)分别为0.81%~0.87%、0.91%~1.5%和0%~0.79%;3-10两性电解质载体、cIEF凝胶、毛细管柱等物料批号改变,方法不受影响。结论建立的cIEF方法专属性、精密度、准确度、耐用性良好,适用于重组假丝酵母尿酸氧化酶pI的检测。

Objective To develop and verify a capillary isoelectrie focusing(cIEF)method for determination of isoelectric point(pI)of recombinant Candida uricase.Methods The cIEF method was developed by optimization of key parameters such as the proportion of stabilizer and focusing time,and verified for specificity,precision,accuracy and durability.Results The optimal ratio of arginine(ARG)to iminodiacetic acid(IDA)in stabilizer was 60μL∶6μL,while the optimal focusing time was 15 min.No interference peak of blank matrix was observed in the position of main peak of recombinant C.uricase.The relative standard deviations(RSDs)of test results of reproducibility,intermediate precision and interbatch precision were 0.81%~0.87%,0.91%~1.5%and 0%~0.79%respectively.The changes of lot numbers of 3-10 amphoteric electrolyte carriers,cIEF gel,capillary column showed no interference to the test results.Conclusion The developed cIEF method showed good specificity,precision,accuracy and durability,which was suitable for determination of pI of recombinant C.uricase.