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目的:建立磷脂化重组人铜锌超氧化物歧化酶(PC-SOD)的活性测定方法,并对方法进行方法学验证。方法:采用经典的氮蓝四唑(NBT)还原法,SOD可竞争结合负氧离子,抑制显色,通过与活性标准品比较抑制显色的程度,计算样品的活性。对方法的专属性、线性与范围、回收率、精密度进行验证。结果:方法符合专属性要求;SOD线性范围0.23~500μg·mL-1,PC-SOD线性范围0.46~1000μg·mL-1,R2≥0.99;回收率测定结果介于80%~120%,符合活性测定方法对于回收率的要求;SOD原液活性测定结果为(5298±549)U·mg-1,PC-SOD原液测定结果为(2765±474)U·mg-1,PC-SOD成品测定结果为(2595±230)U·mg-1,RSD≤20%,符合一般生物制品活性检测精密度要求。结论:建立并验证了反映药效的PC-SOD活性测定方法,可用于PC-SOD的质量控制和一致性分析。
OBJECTIVE: To establish a method for the determination of the activity of phospholipid-induced recombinant human copper-zinc-superoxide dismutase (PC-SOD) and verify the method. Methods: Using classical NBT reduction method, SOD could compete with negative oxygen ions to inhibit the color development. The activity of the sample was calculated by comparing with the active standard to inhibit the color development. The method of specificity, linearity and scope, recovery, precision verification. Results: The method conformed to the requirement of specificity. The linear range of SOD was 0.23-500μg · mL-1, the linear range of PC-SOD was 0.46-1000μg · mL-1, R2≥0.99. The recovery was between 80% and 120% (5298 ± 549) U · mg-1. The result of PC-SOD stock solution was (2765 ± 474) U · mg-1, the result of PC-SOD product was (2595 ± 230) U · mg-1, RSD≤20%, in line with the general biological activity of precision testing requirements. Conclusion: The method of determining the activity of PC-SOD is established and validated, which can be used for the quality control and consistency analysis of PC-SOD.