Human monoclonals against erythrocyte antigens

Abstract

Mice are not able to respond in making specific antibodies against a variety of human antigens. One of these antigens is the erythrocyte D-antigen of the Rh-system. Until now, it has been possible only to make murine monoclonals against structures of the Rh-proteins. These antibodies were not suitable for routine use in blood grouping. As an alternative, the human immune system is able to produce alloantibodies against Rh-D with high specificity. As a result, with the development of reproducible methods for the generation of human monoclonals, we have started to establish protocols for the production of antibodies against the Rh-D antigen. In addition, we selected a heteromyeloma line, MD33, which is a non-secretor line with high fusion rate giving rise to hybridomas which are high producers. Here, we compare different methods for their efficiency in producing human monoclonals against the Rh-D antigen. We tested the direct fusion of human PBL with either mouse myeloma or human lymphoblastoid cell line, immortalization by EBV-transformation, EBV-transformation followed by fusion with mouse myeloma line, and EBV-transformation followed by fusion with heteromyeloma line. We could demonstrate that most success can be achieved by fusions of EBV-transformed PBL with mouse myeloma line P3X63-Ag8.653 and heteromyeloma line MD33 which resulted in several stable and antigen-specific lines per fusion.