Liu Shuangshuang, Li Shasha, Zhang Yang, Wang Ye, Zhu Yumeng, Wang Bin, Chen Zhi-Nan
Department of Cell Biology, National Translational Science Center for Molecular Medicine, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
Mol Med Rep. 2017 Jun;15(6):4035-4040. doi: 10.3892/mmr.2017.6523. Epub 2017 Apr 27.
The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane‑spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen‑immunoaffinity chromatography purification. The purity of rabbit anti‑CD147 polyclonal antibodies was ~99%, and ELISA analysis was able to determine the titer of the rabbit anti‑CD147 polyclonal antibodies at 1:512,000. The lowest concentration of the standard CD147 antigen that the sandwich ELISA was able to detect was 31.25 pg/ml. The sandwich ELISA system was composed of anti‑hepatoma HAb18 monoclonal antibodies and purified rabbit anti‑CD147 polyclonal antibodies. The present study demonstrated that antigen‑immunoaffinity chromatography may be a good technique for the purification of polyclonal antibodies, which may be used to detect antigen in sandwich ELISAs.
免疫球蛋白超家族成员CD147是一种广泛表达的糖蛋白，以跨膜形式和可溶性形式存在。夹心酶联免疫吸附测定（ELISA）是分析可溶性抗原的有力工具。本研究的目的是获得一种针对人CD147的高特异性多克隆抗体，可用于夹心ELISA分析。利用真核表达系统表达重组CD147来免疫兔子以获得抗血清。通过抗原免疫亲和层析纯化获得了一种能够在夹心ELISA中检测可溶性CD147的高特异性多克隆抗体。兔抗CD147多克隆抗体的纯度约为99%，ELISA分析能够确定兔抗CD147多克隆抗体的效价为1:512,000。夹心ELISA能够检测的标准CD147抗原的最低浓度为31.25 pg/ml。夹心ELISA系统由抗肝癌HAb18单克隆抗体和纯化的兔抗CD147多克隆抗体组成。本研究表明，抗原免疫亲和层析可能是一种纯化多克隆抗体的良好技术，可用于夹心ELISA中检测抗原。
