Mol Med Rep. 2017 Jun;15(6):4035-4040. doi: 10.3892/mmr.2017.6523. Epub 2017 Apr 27.

Purification of a polyclonal antibody against CD147 for ELISA using antigen‑immunoaffinity chromatography.

Liu S1, Li S1, Zhang Y1, Wang Y1, Zhu Y1, Wang B1, Chen ZN1.

Author information

1Department of Cell Biology, National Translational Science Center for Molecular Medicine, Fourth Military Medical University, Xi’an, Shaanxi 710032, P.R. China.

Abstract

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.

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