Detection and localization of non-HLA-ABC antigenic sites relevant to kidney rejection on endothelial cells

Immunological rejection of kidney allografts is usually attributed to prese nsitization to HLA antigens. However, data on HLA identical transplant reje ctions indicate that non-HLA antigens may also be involved, and it has been suggested that vascular endothelium represents the main target cell. The p urpose of the present study is to describe a method of detecting non-HLA an tibodies immunocytochemically. We showed the molecular independence between HLA-ABC molecules identified by the monoclonal antibody w6/32, and antigen ic sites identified by a kidney rejection patient serum, previously charact erized, on cultured endothelial cells isolated from human umbilical cords b y collagenase digestion. Single immunofluorescence staining indicated the m olecular independence between these antigenic sites, as the first serum sho wed a granular pattern, diffused throughout the cytoplasm and the other a r eticular pattern restricted to the same cytoplasmic region. This result was confirmed by double labeling. Immunoelectronmicroscopy study also confirme d site independence, showing labeling patterns with different intensities a nd distinct localizations, using 10- and 20-nm colloidal gold particles to reveal HLA-ABC and non-HLA-ABC determinants, respectively. In conclusion, c ultured endothelial cells may be used immunocytochemically to detect non-HL A-ABC determinants of antibody reactivity in renal graft recipients, and th e indirect immunofluorescence may be the methodology of choice, since it is easy, reliable and low cost. (C) 2001 Elsevier Science B.V. All rights res erved.