Generation and characterisation of rabbit monoclonal antibodies against the native cell surface antigens of embryonic stem cells

Abstract

Embryonic stem (ES) cells are potent resources for cell therapy, and monoclonal antibodies (mAbs) against native cell surface markers of ES cells could be useful tools for therapeutic applications. Here, we report the development of a feasible approach, which could be used in mass production, for experimentally producing rabbit mAbs against native cell surface antigens on the cell surface. Two of the 14 mAbs, which were selected at random, could be bound to the cell surface antigens of mES cells. The immunocytochemistry (ICC) and Western blot results showed that mAb 39 recognises conformational epitopes. The target antigen of mAb 39 was then successfully purified using an improved immunoprecipitation approach in which mAb was bounded to intact mES cells before the cells were lysed. The LC-LTQ mass spectrum analysis showed that the target antigen of mAb 39 was Glut3. This result was further confirmed by Western blot using commercially available antibodies against Glut3. Further experiments showed that mAb 39 exhibited an antiproliferative effect on mES cells. We also found that Glut3 was differentially expressed among the mES cell population as detected by flow cytometry.