Development And Preliminary Characterization Of Rabbit Monoclonal Antibody To Vitronectin

The research and application of monoclonal antibodies have already been widely applied to immune analysis, medical diagnosis, vaccine production, gene reconstruction and medicine development. It has become the most active part in the life sciences. Producing monoclonal antibodies using hybridoma technology usually adopt the material of mouse myeloma cell lines. There are always great requirements for rabbit monoclonal antibodies for their high affinity and high specificity.Vitronectin is a multi-functional adhesion protein containing in serum and plasma. The change of vitronectin level is closely related to many kinds of physiology and pathology course, such as blood coagulation, immuno-associated disease, in the human body.In this research, we developed 33 new rabbit-rabbit hybridama cell lines secreting rabbit monoclonal antibodies to purified human protein 'vitronectin' by fusing a new "fusion partner", rabbit myeloma-like cell line of 240E (Epitomics Inc., USA) and rabbit spleen cells immunized by vitronectin, and preliminarily characterized some of these hybridomas. The main research content includes:(1) Cultivation of 240E cellAccording to the drawing of the growth curve, adjust 240E cells growing state and make it suitable for fusion.(2) Immunization of rabbitsUse suitable immunizing method to obtain antisera with high titer and highly activated spleen cells for fusion.(3) Cell fusion and screeningUse PEG to mediate fusion of 240E cells and immunized rabbit spleen cells. Obtain hybridoma cells resisting HAT, and screen by ELISA and Western Blot to get anti-vitronectin positive hybridoma clones.(4) Hybridomas subcloningUse method of 2-dimensional dilution and limited dilution to subclone the hybridoma cells and obtain monoclonal clones by ELISA and Western blot.(5) Preliminary characterization of the hybridomasIncluding 240E cell and hybridoma cell's chromosome analysis, immunohistochemistry analysis, protein across reaction analysis, different epitopes determination, passage stability analysis and ascites induction.