Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cellsReportar como inadecuado




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Biological Procedures Online

, 16:3

First Online: 04 February 2014Received: 29 January 2014Accepted: 01 February 2014DOI: 10.1186-1480-9222-16-3

Cite this article as: Hu, J., Vien, L.T., Xia, X. et al. Biol Proced Online 2014 16: 3. doi:10.1186-1480-9222-16-3

Abstract

BackgroundAlthough genetically engineered cells have been used to generate monoclonal antibodies mAbs against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol GPI-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly.

ResultsWe used Rae-1–overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti–Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining.

ConclusionsOur cell line–based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

KeywordsGPI-anchored protein Rae-1 Monoclonal antibody Hybridomas Streamlined screening strategy Electronic supplementary materialThe online version of this article doi:10.1186-1480-9222-16-3 contains supplementary material, which is available to authorized users.

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Autor: Jiemiao Hu - Long T Vien - Xueqing Xia - Laura Bover - Shulin Li

Fuente: https://link.springer.com/







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