Specific inhibition of HLA class I and II antibodies by soluble antigens--a method for the identification of antibody specificity in sera from alloimmunized individuals

Abstract

HLA class I and II antigens were purified to be used for the determination of the specificity of lymphocyte reactive antibodies in renal transplant patients. Purification of HLA antigens was achieved by affinity chromatography using rabbit antibodies directed to human beta 2 microglobulin, W6/32 antibodies that recognize a nonpolymorphic determinant on HLA class I molecules, and BU25 monoclonal antibodies directed to a monomorphic determinant on HLA class II molecules. Pooled platelets and spleen lymphocytes from a large number of donors were used as a source of class I and II antigens. HLA antigen preparations, highly enriched as shown in Western blot experiments, were obtained that caused a dose-dependent inhibition of the binding as well as of the cytotoxicity of W6/32 and BU25 antibodies. The HLA class I preparation did not inhibit the binding of monoclonal antibodies to T or B cell-specific surface markers or to HLA class II antigens. Similar kinds of results were obtained with the HLA class II antigen preparation, which only specifically blocked the class II antibody binding. The antigen preparations were tested for their ability to block binding of antibodies in sera from alloimmunized patients. In order to avoid complications by soluble HLA antigens, all sera were absorbed in ELISA plates coated with anti-class I and anti-class II monoclonal antibodies. The HLA class I and class II antigen preparations were found to be HLA-specific. All data were compared with the conventional method to determine HLA specificity--i.e., specific blocking of class I and class II reactivity using monoclonal antibodies and unabsorbed sera. Soluble HLA antigens, added directly to mixtures of patient sera and lymphocytes used in the cytotoxicity or binding tests, can therefore be used for determination of the presence of HLA antibodies in sera of alloimmunized patients.