[Preparation and application of mouse-derived monoclonal antibodies against mouse CD226].

Objective To prepare and preliminarily identify anti-mouse CD226 monoclonal antibodies (mAbs) using CD226 knockout (CD226 KO) mice as immunized animals. Methods Animals were immunized by recombinant mouse CD226 protein expressed by eukaryotic cells. Anti-mouse CD226 mAbs were prepared by conventional B-cell hybridoma technology. The application of the generated mAbs for flow cytometry and Western blotting was tested. A sandwich ELISA system was established for the detection of soluble CD226 in mice. And the concentrations of plasma soluble CD226 was determined by this sandwich ELISA system in an LPS-induced sepsis mouse model. Results Two hybridoma cell lines secreting mouse anti-mouse CD226 mAbs were successfully prepared. The clones were named mA1.1 and mA1.3, respectively. The antibody subclasses were both IgG1, and the light chains were κ. The obtained mAbs could be applied for flow cytometry to detect exogenous transfected CD226 on the cell surface and natural CD226 on the mouse platelet membrane. In Western blot assay, the mAb could bind to lymphocyte membrane proteins with a relative molecular mass (Mr) of 67 000 that was consistent with the Mr of CD226. In the sandwich ELISA system, the purified mAbs of mA1.3 were coated on ELISA plates as capture antibody, and the mAbs of mA1.1 were labeled with horseradish peroxidase or biotin as detection antibody. The detection sensitivities were 3.0 and 0.25 ng/mL, respectively. The concentration of plasma soluble CD226 of the septic mice was lower than that of the normal mice in the control group. Conclusion The mouse mAbs for identifying mouse CD226 have been prepared successfully and can be applied in various detection techniques.