Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection
Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection
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DOI：
10.1016/j.chroma.2010.06.072
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发表时间：
2010-08-27
影响因子：
4.1
通讯作者：
Xie, Jianwei
中科院分区：
文献类型：
--
作者：
Shen, Rui;Guo, Lei;Xie, Jianwei
重组人促红细胞生成素-α(rHuEPO-α)已广泛应用于临床治疗贫血。RHuEPO-α的检测和定量是监测这种广泛存在的重组糖蛋白药物的关键。本文首次建立了一种新的亲和探针毛细管电泳法/激光诱导荧光(APCE/LIF)检测rHuEPO-α的方法。在该方法中，通过系统的优化，可以根据核酸适体-rHuEPO-α和游离核酸适体的迁移率和荧光强度很好地分离和鉴定它们的络合物。样品缓冲液和运行缓冲液中钠离子的存在对稳定络合物和提高分离效率起着关键作用，此外，合适的高压和样品缓冲液添加剂也是提高络合物峰高的重要因素。在优化的条件下，该方法成功地用于生理缓冲液、人工尿液和人血清中rHuEPO-α的测定。RHuEPO-α的线性范围为0.2~100 nM，检出限为0.2 nM(即7.4 ng/mL)。进一步的异硫氰酸荧光素标记的rHuEPO-α(F-rHuEPO-α)与N-脱糖基化的F-rHuEPO-α的结合实验表明，寡糖在rHuEPO-α与其适配子的特异性相互作用中起着重要的作用。(C)2010爱思唯尔B.V.保留所有权利。
Recombinant human erythropoietin-alpha (rHuEPO-alpha) has been widely used in clinic for anemia treatment. The detection and quantification of rHuEPO-alpha is essential for monitoring this widespread recombinant glycoprotein pharmaceutical. In this paper, we developed a new affinity probe capillary electrophoresis/laser-induced fluorescence (APCE/LIF) method for the detection of rHuEPO-alpha by using a specific single-stranded DNA aptamer probe for the first time. In this method, the complex of aptamer-rHuEPO-alpha and the free aptamer can be well separated and identified by their migration and fluorescence intensity after systematic optimization. The existence of sodium cation in the sample buffer and running buffer played a critical role for stabilizing complex and enhancing the separation efficiency, additionally, suitable high voltage and sample buffer additives were also important for improving the peak height of the complex. Under the optimized conditions, the method was successfully applied for the quantification of rHuEPO-alpha in physiological buffer, artificial urine and human serum. The linear range for rHuEPO-alpha was from 0.2 to 100 nM and the limit of detection was 0.2 nM (i.e. 7.4 ng/mL). Further binding experiments using fluorescein isothiocyanate-labeled rHuEPO-alpha (F-rHuEPO-alpha) and N-deglycosylated F-rHuEPO-alpha demonstrated that the oligosaccharides moiety was of importance in the specific interaction between rHuEPO-alpha and its aptamer. (C) 2010 Elsevier B.V. All rights reserved.