Human CCDC47 sandwich immunoassay development with electrochemiluminescence technology
Journal of Immunological Methods, 2018, 452:12
Authors: Wenfang S. Wu, Liang Zhu, Saurabh Patil, Karthiga Gokula, Sean Reilly, Joyce Chan, Poornima Tekumalla, Vivek Vishnudas, Michael A.Kiebish, Rangaprasad Sarangarajana, Viatcheslav R. Akmaev, Mark D.Kellogg, Niven R.Narain
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.