Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA.
J Neurosci Methods. 2011 Sep 15;200(2):144-52. doi: 10.1016/j.jneumeth.2011.06.029. Epub 2011 Jul 6.
The transient receptor potential vanilloid receptor type 1 (TRPV1) is a non-selective cation channel expressed in both the peripheral and the central nervous systems. To quantitatively determine TRPV1 protein levels in native rat tissues, novel monoclonal antibodies were raised against full-length recombinant human TRPV1 protein and utilized to develop a sandwich ELISA assay. Monoclonal antibody 10E3-1A2 specifically recognized TRPV1 protein and the recognition epitope was determined to reside in amino acids 45-58 of human and rat TRPV1. Using the TRPV1 polyclonal antibody ABRK4 as the capturing antibody and the monoclonal antibody 10E3-1A2 as the detection antibody, a sandwich ELISA that detected both human and rat TRPV1 protein was established. Recombinant human TRPV1 heterologously expressed in mammalian HEK293-F cells, which showed high ligand-binding affinity, was purified by TRPV1 monoclonal antibody affinity chromatography and used as protein standard to quantify TRPV1 protein levels. This ELISA detected TRPV1 protein as low as 1.5ng/ml (15pM), and was able to determine TRPV1 protein levels in native rat tissues such as DRG and spinal cord. This is the first TRPV1 sandwich ELISA that determines the abundance of TRPV1 protein in different tissues. It provides a powerful tool to quantify changes of TRPV1 protein levels in pathological states.
瞬时受体电位香草酸受体 1 型（TRPV1）是一种非选择性阳离子通道，在周围神经系统和中枢神经系统中均有表达。为了定量测定天然大鼠组织中的 TRPV1 蛋白水平，我们针对全长重组人 TRPV1 蛋白产生了新型单克隆抗体，并将其用于开发夹心 ELISA 检测法。单克隆抗体 10E3-1A2 特异性识别 TRPV1 蛋白，其识别表位位于人源和大鼠 TRPV1 的 45-58 位氨基酸。使用 TRPV1 多克隆抗体 ABRK4 作为捕获抗体，以及单克隆抗体 10E3-1A2 作为检测抗体，建立了可同时检测人源和大鼠 TRPV1 蛋白的夹心 ELISA。通过 TRPV1 单克隆抗体亲和层析，从表达具有高配体结合亲和力的人源 TRPV1 的哺乳动物 HEK293-F 细胞中纯化重组人 TRPV1，并将其用作蛋白标准来定量 TRPV1 蛋白水平。该 ELISA 最低可检测到 1.5ng/ml（15pM）的 TRPV1 蛋白，并且能够测定 DRG 和脊髓等天然大鼠组织中的 TRPV1 蛋白水平。这是首个测定不同组织中 TRPV1 蛋白丰度的 TRPV1 夹心 ELISA，为定量检测病理状态下 TRPV1 蛋白水平的变化提供了有力工具。
