Preparation Of Monoclonal Antibody Against Human Ospteopontin And Analysis Of Its Specifity

Osteopontin (OPN:also called'early T cell-activation gene 1') is a multifunctional protein that has been linked to many physiological and pathological events, including bone remodeling, cancer and inflammation. The much higher expression of OPN at the site of pathology in several autoimmune diseases, including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel disease, has focused attention on this molecule as a potentially critical factor in pathogenesis. T cell-mediated inflammation plays an important role in autoimmune diseases. The binding of OPN to a variety of cell surface receptors induces signaling events that promote cell adhesion, migration and survival. OPN has different functional domains and each functional domain results in different functions by interaction with unidentical receptors. So far, the physiologic or patho-physiologic roles of the far N-terminal osteopontin domains (including exons 4 and 5) have not yet been defined.Objective:To identify novel function of OPN,we prepared monoclonal antibody against ospteopontin.Methods:BABL/c mice were immunized with recombinant human ospteopontin (rhOPN) and splenocytes of the immunized mice were fused with myeloma NS1 cells to produce hybridoma cell lines. ELISA assay was used to identify the monoclonal antibody against ospteopontin protein and Western blotting were applied to identify the specificity of this antibody.Results:It was demonstrated that 2 monoclonal antibodies recognizing 2 different epitopes on OPN were obtained. These antibodies showed specific binding power to rhOPN. The subtype of 2 antibodies was proved to be IgG1. These antibodies showed specific binding activity to rhOPN. It is evident that monoclonal antibodies against OPN-mediated cells migration.conclusions:the anti-OPN monoclonal antibodies may provide asolid basis for the further studies on the biological characteristics of ospteopontin.