Monoclonal antibodies to normal and abnormal epithelial antigens

Mouse monoclonal antibodies to various human epidermal and basement membrane components were formed by immunizing Balb/c mice with ME-180, a line of human cervical carcinoma cells. The spleen cells from hyperimmunized mice were fused with a nonsecreting mouse myeloma cell line using polyethylene glycol. The resulting hybrids were selected by growth in media containing 20% fetal calf serum, hypoxanthine, thymidine, and methotrexate in RPMI-1640 in 24-well Linbro plates. Wells producing antibodies of interest were grown and eventually cloned over an HGPRT- rat fibroblast feeder layer. These cultures were expanded and recloned. Two cloned antibodies of interest are DUX 5.2 and DUX 1.1.3. DUX 5.2 is the mouse IgG1 subclass and reacts with the membranes of ME-180 cells and the human skin epidermal basement membrane zone as shown by direct immunofluorescent microscopy. Ultrastructural localization using electron microscopic immunoperoxidase techniques showed localization of the DUX 5.2 antigen to be beneath the lamina densa; the reaction product may include the anchoring fibrils. Although DUX 5.2 reacts with the normal human basement membrane zone and the basement membrane zone in several diseases, there is no reactivity in the normal, never-blistered skin of patients with dystrophic epidermolysis bullosa (DEB). This suggests that the increased collagenase in the disease may be destroying antigenicity of the antigen recognized by DUX 5.2 or that the antigen may not be present in DEB. This antibody will thus allow early neonatal and prenatal diagnosis in DEB and allow isolation of the structural moiety which is deficient in DEB. DUX 1.1 is an IgM mouse immunoglobulin specific for the cytoplasm of human basal cells. Its reactivity with upper epidermis is significantly less than that seen in the basal layer. All cells of the basal layer stain uniformly. The slight amount of staining in upper cells probably represents dilution of antigen which is not synthesized beyond the basal layer. Basal cells of hair follicles and sweat glands are stained to some degree.