Li Nan, Cook Lillian, Santos Cheryl, Cass Carol E, Mackey John R, Dovichi Norman J
Department of Chemistry, University of Alberta, Edmonton, Canada.
Anal Chem. 2002 Jun 1;74(11):2573-7. doi: 10.1021/ac025559r.
The human equilibrative nucleoside transporter 1 protein (hENT1) is a major mediator of cellular entry of nucleosides and anticancer nucleoside drugs; its assay is important in understanding and diagnosing chemotherapy resistance. Here we present a novel assay for quantifying hENT1 using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A cellular population is treated with 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine-x8-fluorescein (5-SAENTA-x8f), which binds with high affinity and specificity to the hENT1 protein. The cells are washed to remove excess reagent, lysed, and centrifuged, and the supernatant is analyzed by CE-LIF with the use of an internal standard. Accuracy was evaluated by comparing the capillary electrophoresis results with those obtained by flow cytometry; the results were highly correlated, r = 0.96. The relative standard deviation of the hENT1 assay was 10%, determined from nine independent assays of the same cell line, which is 3 times superior to results obtained in a flow cytometry assay. The detection limit for 5-SAENTA-x8f was 4300 molecules injected into the capillary.
人平衡核苷转运体1蛋白（hENT1）是核苷和抗癌核苷药物进入细胞的主要介导因子；其检测对于理解和诊断化疗耐药性很重要。在此，我们提出一种使用毛细管电泳-激光诱导荧光检测（CE-LIF）定量hENT1的新方法。用5'-S-（2-氨基乙基）-N6-（4-硝基苄基）-5'-硫代腺苷-x8-荧光素（5-SAENTA-x8f）处理细胞群体，该物质与hENT1蛋白具有高亲和力和特异性结合。洗涤细胞以去除多余试剂，进行裂解和离心，然后使用内标通过CE-LIF分析上清液。通过将毛细管电泳结果与流式细胞术获得的结果进行比较来评估准确性；结果高度相关，r = 0.96。hENT1检测的相对标准偏差为10%，由对同一细胞系的九次独立检测确定，这比流式细胞术检测结果优3倍。5-SAENTA-x8f的检测限为注入毛细管中的4300个分子。
