PRODUCTION OF STABLE RABBIT MOUSE HETEROHYBRIDOMAS - CHARACTERIZATIONOF A RABBIT MONOCLONAL-ANTIBODY RECOGNIZING A 180-KDA HUMAN LYMPHOCYTE MEMBRANE ANTIGEN

Polyclonal rabbit antihuman thymocyte globulin (RATG) remains a key co mponent of immunosuppressive strategies in transplantation. The human thymus immunization regimen that produces highly immunosuppressive RAT G induces unique antibody specificities in the rabbit. Rabbit monoclon al 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 i mmunized with human thymus and have identified 52 rabbit-mouse heteroh ybridomas which secrete RAb MAbs directed against human lymphocyte sur face antigens. The technical aspects of hybridoma isolation, stabiliza tion and characterization are presented. Analysis by flow cytometry, p reabsorption and immunoprecipitation suggests that RAb MAb 1A8 IgG may recognize LFA-1, one of the principal lymphocyte surface antigens rec ognized 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-e xpression in two-color FACS analysis using four different murine anti- LFA-1 MAbs. 1A8 markedly inhibits the mitogenic response of lymphocyte s to PHA, as do murine anti-LFA-1 MAbs. A combination of rabbit antily mphocyte MAbs may potentially reproduce the multiple specificities fou nd in polyclonal RATG and lead to the production of a superior immunos uppressive clinical agent.