The objective of this project is to produce and characterize some murine monoclonal antibodies against specific markers in different types of retinal cells in adult rabbit. The hybridomas raised will be used as consistent and cost-effective sources of the specific monoclonal antibodies for future development as commercial supplies. These antibodies can be widely used as valuable molecular probes in the studies of the identification, classification, cell lineage cellular interaction and differentiation of different types of mammalian retinal cells. In this proposed study, BALB/c mice will be first immunized in vivo with either crude retinal membranes or partially purified dissociated retinal cells. The immune spleen cells will be subjected to 4 days in vitro immunization with these immunogens. The lymphocytes are then fused with myeloma cells (P3 x 63-Ag8.653); the hybridomas will be isolated by HAT medium and cloned. The monoclonal antibodies are screened by immunocytochemistry on retinal section or isolated retinal cells for both intracellular or cell-surface markers. The specific monoclonal antibodies will be produced from ascites fluid by injecting hybridomas into BALB/c mice, and purified by hydroxylapatite column chromatography. The specific hybridoma cells will be frozen as permanent sources of the antibodies.
