脑脊液中微量的可溶型晚期糖基化终末产物受体检测方法建立与初步应用

Detection of Trace Amount Soluble Receptor for Advanced Glycation End-productin in Human CSF

目的利用电化学和纳米金颗粒相结合的技术检测脑脊液中的可溶型晚期糖基化终末产物受体(sRAGE)表达水平。方法依次利用肝素和特异性抗体的层析柱纯化sRAGE蛋白,把纯化的蛋白作为标准品检测电化学和纳米金颗粒相结合技术的准确性和灵敏度,建立稳定的检测方法后,检查脑脊液中sRAGE的表达。结果重组sRAGE蛋白经2次纯化后,纯度超过95%。利用电化学和纳米金颗粒相结合的技术检测标准品的标准曲线,其R2值达到了0.96。利用其检测脑脊液样本证明sRAGE确实在脑脊液中表达,其表达量在pg水平。结论sRAGE存在于脑脊液中,电化学和纳米金颗粒相结合的技术可作为更灵敏的检测方法应用于临床及科研中。

Objective Using the combination of Au nanoparticles and electrochemistry method to detect trace amount soluble receptor for advanced glycation end-product （sILAGE） in human cerebrospinal fluid （CSF）. Methods Purify sRAGE protein from conditioned medium of cultured 293T cell overexpressing sRAGE with heparin and specific antibody column, electrochemistry measurement was applied to detect the reduction potential of Au nanoparticle which was specifically bound to sRAGE. Standard method was established and applied to measure the concentration of sRAGE in CSF. Results The purity of purified sRAGE protein was more than 95%. The R2 value obtained by elec-trochemistry measurement using sRAGE standard protein was 0.96. sRAGE was detected in human CSF, and its expression is at pg level. Conclusion sRAGE is actually expressed in human CSF,and the combination of Au nanoparticles and electrochemistry method is more sensitive to be used to detect trace amount protein.