A mouse monoclonal antibody specific for calreticulin.

Calreticulin (CRT) has various versatile functions. It is one of the family of heat shock proteins (HSPs), and is mainly located in the lumen of the endoplasmic reticulum (ER). In order to elucidate the functional and structural properties of CRT, we expressed and purified CRT protein; we then developed a monoclonal antibody (MAb) against mouse CRT by immunizing BALB/c mice with a specific region of the N and P domains of CRT (dCRT) as antigen, which was expressed in Escherichia coli. A stable hybridoma cell line was established by enzyme linked immunosorbent assay (ELISA) screening. The MAb was then prepared from mouse ascites after inoculating the hybridoma cells. Different methods were used to analyze the characterization of the MAb: ELISA, Western blot analysis, immunofluorescence, and flow cytometry. The dCRT protein was expressed and purified and a MAb cell line for CRT was established through immunization, fusion, and screening. ELISA and Western blot analysis indicated that the MAb specifically recognized CRT. In addition, immunofluorescence and flow cytometry demonstrated that the MAb exhibits excellent reactivity to the ecto-CRT when the cells were induced to apoptosis. This CRT MAb will be a valuable tool for further investigation of calreticulin functions.