Wu Wenfang S, Zhu Liang, Patil Saurabh, Gokul Karthiga, Reilly Sean, Chan Joyce, Tekumalla Poornima, Vishnudas Vivek, Kiebish Michael A, Sarangarajan Rangaprasad, Akmaev Viatcheslav R, Kellogg Mark D, Narain Niven R
BERG, LLC, 500 Old Connecticut Path, Framingham, MA 01701, USA; Department of Pathology, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
BERG, LLC, 500 Old Connecticut Path, Framingham, MA 01701, USA.
J Immunol Methods. 2018 Jan;452:12-19. doi: 10.1016/j.jim.2017.09.011. Epub 2017 Sep 30.
Coiled-Coil Domain Containing 47 (CCDC47) is an endoplasmic reticulum (ER) transmembrane protein involved in calcium signaling through utilization of its calcium binding-acidic luminal domain. CCDC47 also interacts with ERAD (endoplasmic reticulum-associated degradation) complex and is involved in ER stress relief. In this report, we developed human CCDC47 monoclonal antibodies and a sandwich immunoassay for CCDC47 measurement in biological matrices. Specificity of developed antibodies were confirmed by immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated cell lysates. To achieve high analytical sensitivity, traditional colorimetric enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence (ECL) technology were compared, and 3 logs of increased sensitivity was observed with the use of ECL. A CCDC47 sandwich ECL assay was subsequently developed and performances evaluated for calibration curves, precision and accuracy, as well as selectivity and interferences for sample measurement. Sample stability was also characterized for freeze/thaw cycles and short/long term storage conditions.
卷曲螺旋结构域蛋白47（CCDC47）是一种内质网（ER）跨膜蛋白，通过利用其钙结合酸性腔结构域参与钙信号传导。CCDC47还与内质网相关降解（ERAD）复合物相互作用，并参与内质网应激缓解。在本报告中，我们开发了人CCDC47单克隆抗体和用于在生物基质中测量CCDC47的夹心免疫测定法。通过免疫印迹和免疫沉淀细胞裂解物的液相色谱-串联质谱（LC-MS/MS）分析确认了所开发抗体的特异性。为了实现高分析灵敏度，比较了传统的比色酶联免疫吸附测定（ELISA）和电化学发光（ECL）技术，使用ECL观察到灵敏度提高了3个对数。随后开发了CCDC47夹心ECL测定法，并对校准曲线、精密度和准确度以及样品测量的选择性和干扰进行了性能评估。还对冻融循环和短期/长期储存条件下的样品稳定性进行了表征。
