Intrasplenic immunization for production of monoclonal antibodies against mouse blastocysts.

Applying the intrasplenic immunization method monoclonal antibodies were raised against trophectoderm of mouse blastocysts. Adhesive C57BL/6 blastocysts, obtained 18 h after estrogen reactivation from an experimental delay of implantation, and irradiated with 5000 rad were used as immunogen. Male DBA/2 mice were immunized by four intrasplenic depositions of about ten blastocysts each. The sensitized spleen cells were fused with mouse plasmacytoma cells on the 5th day after the last booster, followed by isolation of hybridoma clones by conventional monoclonal antibody procedures. 82 hybridoma clones were obtained of which two produced IgM antibodies recognizing trophoblast determinants. Absorbing the monoclonal antibodies with C57BL/6 splenic leukocytes followed by immunolabelling of blastocysts demonstrated that the antibodies recognized neither MHC nor TLX antigens. Pre- and peri-implantation stages were mapped by indirect immunofluorescence microscopy. Morulae were negative while blastocysts were positively labeled. Adhesive blastocysts labeled more strongly than delayed blastocysts. Cultured blastocysts showed an intense labeling of some of the trophoblast cells, while other trophoblast cells were unlabeled.