Development and characterization of monoclonal antibodies directed against the retinal pigment epithelial cell

Abstract

The retinal pigment epithelium consists of a unicellular layer of neuroepithelial cells that are essential for the maintenance of normal function of the neural retina. In order to evaluate more critically this cell in health and disease, we prepared monoclonal antibodies against human retinal pigment epithelial (RPE) cells. Balb/c mice were immunized with human RPE cells. Spleen cells were fused with myeloma cells and resultant hybridomas were selected for antibody production. Supernatants were assayed by immunoperoxidase on frozen sections of human eye tissues. Two hybrids were cloned and ascites were generated in mice. These IgG antibodies react only with RPE cells and show no cross-reactivity with other cells in the eye or with human brain, kidney, skin, salivary glands, lymphocytes or monocytes. These antibodies recognize cell surface molecules that are highly conserved since they can be found in man, monkey, rat, cow, chicken and frog. SDS gel electrophoresis and immunoblot analysis showed that one of the antibodies reacted with a 42,000 MW polypeptide. Evaluation of the developing rat retina revealed that the epitopes are not detected at birth, are weakly present at day 6 and are highly recognized by day 9. These immunoglobulins will allow us to evaluate RPE cells in disease (proliferation, migration) and to probe the bioregulatory functions (phagocytosis, vitamin A transport) of these cells.