建立和优化重组人源中期因子(recombinant human midkine，rhMK)活性检测方法，并对其方法进行验证。本文采用细胞计数试剂盒-8(cell counting kit-8，cck-8)法检测rhMK作用大鼠膝软骨细胞的促增殖活性，并对方法进行专属性、准确性、精密性、线性、耐用性验证。RhMK的缓冲液及重组人源白介素-1受体拮抗剂无明显活性效力，该方法专属性良好;相对活性为50％、75％、100％、125％、150％的样品效价回收率(n=6)统计在80。0％～124。0％之间，各相对效价的相对标准偏差均在20％以内，线性相关系数R2≥0。98，表明方法的准确性、精密性、线性均良好;耐用性相关系数R2 ≥ 0。92，量效曲线的最大值与最小值的比值≥1。5，方法的耐用性良好。研究建立的rhMK的生物学活性检测方法，其方法验证良好，为rhMK开发过程中常规样品的生物学活性分析提供了可靠的检测方法。本研究已获得上海南方模式生物科技股份有限公司动物看护与使用委员会批准(批准号:2019-0008-06)。
Establishment and validation of bioactivity measurement method for recombinant human midkine
To establish and optimize a method for the detection of recombinant human midkine(rhMK)activity and verify its methodology,cell counting kit-8(cck-8)method was used to measure the proliferation activity of rat knee chondrocytes.The specificity,accuracy,precision,linearity and robustness of the method were also verified in this study.The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect;the recoveries of the samples with relative activities of 50％,75％,100％,125％,150％were in the range of 80.0％to 124.0％by statistical analysis,the relative standard deviations(RSD)of relative potency were all within 20％,the linear correlation coefficient,R2 ≥ 0.98,suggesting that the accuracy,precision and linearity of the method were good;the robustness correlation coefficient,R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5,indicating that robustness of the methods was good.In conclusion,a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development.This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center,Inc.(approval number:2019-0008-06).
recombinant human midkinecck-8bioactivityproliferationmethod validation
万方数据
维普
