Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.
Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.
复制标题
DOI：
10.1039/c5an02468d
复制
发表时间：
2016-03-21
期刊：
影响因子：
--
通讯作者：
Roper MG
中科院分区：
文献类型：
--
作者：
Yi L;Wang X;Bethge L;Klussmann S;Roper MG
证明了使用荧光标记的镜像适配体（所谓的Spiegelmers）检测胰高血糖素和胰淀素的皮摩尔浓度的能力。Spiegelmers可与抗体的特异性相媲美，并克服了生物基质中天然适体的生物稳定性问题。以Spiegelmers为亲和探针，建立了胰高血糖素和胰淀素的非竞争性毛细管电泳亲和分析方法。胰高血糖素的检测限为6 pM，胰淀素的检测限为40 pM。胰高血糖素样肽-1和-2不干扰胰高血糖素测定，而胰淀素测定显示与降钙素基因相关肽交叉反应。将开发的测定与胰岛素的竞争性免疫测定结合，以测量在与不同葡萄糖浓度孵育后来自批次胰岛的胰高血糖素、胰淀素和胰岛素分泌。这些检测的发展是一个重要的一步纳入在线测量系统监测动态分泌从单个胰岛。
The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.