Isolation and characterization of a mouse monoclonal antibody against human corneal endothelial cells

In vitro cultivation of human corneal endothelial cells (HCEC) is associated with loss of typical cobblestone-like appearance during successive passages. Thus far morphology was the sole criterion for the cell's endothelial nature. Mouse monoclonal antibodies (mabs) to human corneal endothelial cells were raised using standard immunization and hybridoma isolation procedures. The specificity of mabs for human corneal endothelial cells was tested in comparison to other endothelial cell types, to fibroblasts, corneal keratocytes and to human retinal pigmented epithelial cells. In addition immunofluorescence or immunoperoxidase staining was performed with frozen tissue sections of human corneas and with various other human tissues. The mab 9.3.E reacts with cultured human corneal endothelial cells, but not with cultured human fibroblasts and human keratocytes. In frozen sections selective positivity of corneal endothelium in contrast to negativity of the other corneal cell types was confirmed. In investigated extraocular tissues positivity was observed in smooth muscle cells including related cells (i.e. Ito and mesangial cells) and in Schwann's cells and adipocytes, but apparently not in vascular endothelial cells. The mab is human-specific and binds to a protein with a molecular weight of 130 kDa mainly accumulating along cell membranes. A mouse monoclonal antibody against human corneal endothelial cells was established in vitro and was shown to be capable of differentiating corneal endothelial cells from other corneal cell types, especially from corneal keratocytes. It is, however, not cornea-specific, but also reacts with certain extraocular cell types.