German I, Roper M G, Kalra S P, Rhinehart E, Kennedy R T
Department of Chemistry, University of Florida, Gainesville, USA.
Electrophoresis. 2001 Oct;22(17):3659-67. doi: 10.1002/1522-2683(200109)22:17<3659::AID-ELPS3659>3.0.CO;2-I.
A competitive immunoassay for neuropeptide Y (NPY) based on capillary electrophoresis (CE) with laser-induced fluorescence detection was developed utilizing polyclonal antisera as the immunoreagent and fluorescein-labeled NPY as the tracer. The assay was performed with on-line mixing of reagents, automated injections, and a 3 s separation time. The assay had a detection limit of 850 pM. To detect NPY at lower concentrations, the assay was coupled on-line to reversed-phase capillary liquid chromatography (LC). In this arrangement, 5 microL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol-containing mobile phase. The resulting chromatographic peaks were monitored by the CE immunoassay. With preconcentration, the concentration detection limit was improved to 40 microM and NPY could be measured in push-pull perfusion samples collected from the paraventricular nucleus of freely moving rats. The technique was extended to simultaneous detection of NPY and glucagon secretion from islets of Langerhans.
基于毛细管电泳（CE）和激光诱导荧光检测技术，开发了一种用于检测神经肽Y（NPY）的竞争性免疫分析方法。该方法使用多克隆抗血清作为免疫试剂，荧光素标记的NPY作为示踪剂。通过试剂在线混合、自动进样以及3秒的分离时间来进行分析。该分析方法的检测限为850 pM。为了检测更低浓度的NPY，该分析方法与反相毛细管液相色谱（LC）进行在线联用。在这种配置下，5微升样品通过毛细管LC进行预浓缩，并通过含异丙醇的流动相梯度洗脱。所得色谱峰通过CE免疫分析进行监测。通过预浓缩，浓度检测限提高到了40 microM，并且可以在从自由活动大鼠的室旁核采集的推挽灌注样品中检测NPY。该技术还扩展到了同时检测胰岛中NPY和胰高血糖素的分泌。
