PRODUCTION OF STABLE RABBIT MOUSE HETEROHYBRIDOMAS - CHARACTERIZATIONOF A RABBIT MONOCLONAL-ANTIBODY RECOGNIZING A 180-KDA HUMAN LYMPHOCYTE MEMBRANE ANTIGEN
Km. Verbanac et al., PRODUCTION OF STABLE RABBIT MOUSE HETEROHYBRIDOMAS - CHARACTERIZATIONOF A RABBIT MONOCLONAL-ANTIBODY RECOGNIZING A 180-KDA HUMAN LYMPHOCYTE MEMBRANE ANTIGEN, Hybridoma, 12(3), 1993, pp. 285-295
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.