血浆中游离甲氧基肾上腺素类物质化学发光免疫检测方法的建立及性能确认

Establishment and verification validation of plasma free metanephrines measurement by chemiluminescence immunoassay

目的建立血浆中游离甲氧基肾上腺素类物质的化学发光免疫检测方法并对检测方法部分性能进行确认。方法将待测样品(校准品、血浆或质控品)进行萃取、酰化,处理后的待测物分别与固相化甲氧基肾上腺素(MN)、甲氧基去甲肾上腺素(NMN)的特异性抗原竞争结合辣根过氧化物酶(HRP)标记的特异性抗体,通过免疫反应,形成固相化的抗原-抗体-HRP免疫反应复合物,HRP催化底物发光。用特异性、灵敏度、精密度、热加速稳定性和准确度进行性能确认。结果建立的MN、NMN检测方法检测多种结构类似物均无明显的交叉反应;MN检测试剂的空白限、检出限、功能灵敏度分别为10.51、21.15、25.76 ng/L,NMN检测试剂分别为11.54、28.43、31.29 ng/L;MN、NMN检测试剂变异系数分别为2.41%~5.38%,1.61%~3.22%;2种检测试剂的热加速稳定性试验显示检测试剂在2~8℃可以稳定存放1年,且该方法量值结果可溯源至质谱。结论本研究成功建立了检测血浆中游离MN、NMN的化学发光免疫检测方法。

Objective To establish a chemiluminescence immunoassay method for free metanephrines measurement in plasma,and verify its performance.Methods The samples(calibrator,plasma,and quality control sample)were extracted and acylated,the processed analyte samples were then competed with the immobilized specific antigens of metanephrine(MN),normetanephrine(NMN)respectively,to combine specific antibodies marked by Horseradish Peroxidase(HRP),the immobilized antigens-antibody-HRP immune reaction product was then formed through the immune reaction,which could be detected through measurement of HRP catalytic substrate luminescence.Performance verification was performed using specificity,sensitivity,precision,thermally accelerated stability,and accuracy metrics.Results The established detection methods for MN and NMN have no obvious cross-reactions when detecting multiple structural analogs;the limit of blank,limit of detection and functional sensitivity of the MN detection reagent was 10.51,21.15 and 25.76 ng/L,and the performance of the NMN detection reagent was 11.54,28.43 and 31.29 ng/L;the coefficients of variation of the detection reagents for MN and NMN were 2.41%-5.38%and 1.61%-3.22%respectively;the accelerated stability test of the two detection reagents showed that the reagents could be stored stably for 1 year at 2-8℃;and the measurement results of this method can be traced to the mass spectrometer.Conclusion In this study,a chemiluminescence immunoassay detection method for free metanephrines in plasma is successfully established.