Production of stable rabbit-mouse heterohybridomas: characterization of a rabbit monoclonal antibody recognizing a 180 kDa human lymphocyte membrane antigen.
Polyclonal rabbit antihuman thymocyte globulin (RATG) remains a key component of immunosuppressive strategies in transplantation. The human thymus immunization regimen that produces highly immunosuppressive RATG induces unique antibody specificities in the rabbit. Rabbit monoclonal antibodies (RAb MAbs) to human T cell antigens would be of value in the effort to investigate and reproduce the multiple specificities of RATG. We have fused mouse Sp2/0 cells with splenocytes from rabbits immunized with human thymus and have identified 52 rabbit-mouse heterohybridomas which secrete RAb MAbs directed against human lymphocyte surface antigens. The technical aspects of hybridoma isolation, stabilization and characterization are presented. Analysis by flow cytometry, preabsorption and immunoprecipitation suggests that RAb MAb 1A8 IgG may recognize LFA-1, one of the principal lymphocyte surface antigens recognized by RATG. The 1A8 antigen is 180 kDa and is expressed by 80-90% human PBL and thymocytes. LFA-1 and the 1A8 antigen exhibit 100% co-expression in two-color FACS analysis using four different murine anti-LFA-1 MAbs. 1A8 markedly inhibits the mitogenic response of lymphocytes to PHA, as do murine anti-LFA-1 MAbs. A combination of rabbit antilymphocyte MAbs may potentially reproduce the multiple specificities found in polyclonal RATG and lead to the production of a superior immunosuppressive clinical agent.