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Published on: July 5, 2018
[Preparation and characterization of monoclonal antibodies against human CD112 (Nectin2/PRR2)]
Hu-Ping Deng1, Ran Zhuang, Chao-Jun Song
1Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
Aim:
To express and purify fusion protein of human CD112 extracellular region, prepare monoclonal antibodies (mAb) to CD112 and investigate the distribution of CD112 molecule.
Methods:
The CD112-Fc fusion protein was expressed with gene recombinant technique in eukaryotic system and purified with affinity chromatography column. The BALB/c mice were immunized with purified CD112-Fc for preparation of mAb by hybridoma technique. The prepared mAbs were identified by indirect ELISA, Western blot and flow cytometry (FCM). Subsequently, the distribution of CD112 on different human cell lines was investigated by FCM.
Results:
The effective expression plasmid pIg3C-CD112 was constructed, and human CD112-Fc was successfully expressed and purified with a purity of more than 90%. Nine clones of hybridoma were obtained, which were designated as FMU-CD112.1-FMU-CD112.9. FMU-CD112.1, 3, 6 and 8 specifically reacted with recombinant CD112-Fc protein in Western blot and FMU-CD112.1, 4, 6 and 8 could be used in FCM. The investigation of CD112 distribution showed high expression on the cell lines differentiated from epithelial cells.
Conclusion:
The hybridomas secreting mAbs to human CD112 are established successfully, which may lay the foundation for further research on the biological function of CD112.
