Upsala J Med Sci 88: 151-153, 1983 Experiments with Immunization of Mice with Blastocysts by an Intrasplenic Route B . Ove Nilsson,' K . - 0 . Gronvik2 and P. C. Svalander' Departments of 'Human Anatomy and 2Zmmunology, Biomedical Centre, Vppsala, Sweden ABSTRACT A few attechirig blastocysts from CEiAIH mice were irradiated from a Cesium source and transferred into the spleen of male D B A 2 mice. A booster immunization was performed after four weeks. Blood samples for preparation of antiserum to test for the presence of immunoglobulins directed against blastocyst surface determinants were obtained by a retro-orbital puncture. Specific antibodies were detected with a protein A-gold method, modified for transmission electron microscopy of air-dried blastocysts. The results showed that C B A I H blastocyst incubated in D B A 2 immune serum were positive for protein A-gold labelling, while control blastocysts only possessed a few irregularily scattered gold particles. T h u s , it seems a s a deposition of antigens in the spleen tissue with persistence of the antigens at this site will result in detectable afitibodies in the peripheral blood. INTRODUCTION Raising antiserum hgsinst blastocyst antigens is laborious, since a con- ventional immunization requires thousands of blastocysts ( 1 , 4 , 6 ) . A more rational way could be the use of a spleen cell culture, that i s , a few blasto- cysts ( t h e antigens) are co-cultured with spleen cells which then are used to produce monoclonal antibodies ( 2 ) . In the present report we describe experi- ments with a technique where the blastocysts were deposited directly into the spleen tissue, where they seemed capable to trigger an antibody response. MATERIAL ANC METHGDS Blastocysts were obtained from C B A / H mice in delayed implantation. The blastocysts were recovered 18 h a f t e r an injection of oestrogen, that i s , when they were about to attach onto the uterine surface. The flushings were made with PBS containing 1 p e r cent serum from the future host o r 5 0 0 IEIml hepa- rin. Since GUP prime goal was to obtain antibodies against surface constituents 151 of attaching trophoblast cells, the blastocysts were irradiated, before t h e transfer into the spleen, with 5000 R from a Cesium source to block their ability to grow. Recipients were 4 male mice, aged two months, of the DEA2 strain. The spleen was reached through a dorso-lateral incision, and the caudal end of the spleen was carefully taker. out of the abdominal cavity. A micropipette containing 2-3 donor blastocysts in a small amount of PBS with recipient serum o r heparin was inserted underneath the splenic capsule, and the blastocysts were extruded from the pipette. The fluid remaining in the pipette was examined in o r d e r to determine whether all of t h e blastocysts had been extruded. A f t e r a successful transfer the spleen was replaced in position, arid the incision was closed. After four weeks, a booster inimunization was performed by transfer of sin;ilsrily treated blastocysts. Between one and three weeks later, blood samples were taken frcm each recipient animal by retro- -orbital puncture, and serum was prepared for testing for the presence of immunoglobulins directed against trophcblast determinants. The presence of specific antibodies was detected with a protein A-gold method (3,5) modified for transmission electron microscopy of blastocysts (Svalander, Ljung arid Nilsson, in preparation). Blastocysts in the same functional state as those used for immunization were flushed from the uterine horns of C B A I H mice with a fixative of 0 . 5 % glutaraldehyde in PDS into a watchglass. The blastocysts were left in the fixative for a maximum of 20 rnin, washed four times in P S S and incubated in the DBA2 immune serum diluted 1:20 for 45 min at room temperature. Controls were incubated in PBS or in DEA2 non-immune serum. After three subsequent washings in P B S , the blastocysts were incubated in rabbit anti-mouse-Ig antiserum diluted 1: 5G for 30 min at room temperature, and washed three times in PBS. The blastocysts were then transferred to the protein A-colloidal gold solution, in which they were left for 30 min at r o o m temperature. A f t e r this labelling, the blastocysts were washed three times in PBS and twice in double-distilled, ultrafiltered water. Finally, they were placed on a hexagonal grid ( h e x 700 T B , Polaron Eq. Ltd., England) without membrane in a microdroplet of water, and were left to d r y in the air. The air-dried blastocysts were examined in a Philips 400 STEM electron microscope with field emission gun operated at 100 kV to evaluate the number of gold particles. RESULTS A N D COMkENTS Blastocysts flushed from CBA/H mice 18 h after an injection of estrogen and incubated in D B A 2 immune sera were positive for protein A-gold labelling. The particles observed in the micrographs imaged gold colloid from both the upper and lower surface of the flat blastocyst. The particles were 152 irregularily scattered in the field of view. The control blastocysts, which were incubated in PBS o r in D B A 2 non-immune serum instead of the immune serum at the first incubation s t e p , possessed only a few irregularily scattered gold particles. T h u s , it seems a s B deposition of antigens in the spleen tissue with persistence of the antigens at this site will result in a detectable amount of antibody in the peripheral blood. This preliminary finding is now f u r t h e r tested using, among other things, gel plugs with bovine serum albumin a s antigen. R E FEREN CE S 1. Johnson, L. V . & Calarco, P. G . : Stage-specific embryonic antigens detected by an antiserum against nouse blastocysts. Dev Eiol 7 9 : 224-231, 1980. antibodies to hypothalamic growth hormone-releasing factor with picornoles of antigen. Science 218 : 887-889, 1 9 8 % . quantitative approach for antigen localizatior, on thin sections. In: Techniques in Immuriocytochemistry (eds. G . R . Bullock & P. Petrusz), pp. 107-133. Academic Press, London, 1982. 4 . Shevinsky, L . H . , Knowles, B.B., Damjanov I . & Solter, D . : Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. Cell 2 . Luben, R.A., Brazeau, P . , Bohlen, P. & Guillemin, R . : Monoclonal 3 . Roth, J.: The protein A-gold (pAg) technique. Qualitative and 3 0 : 6 9 7 - 7 0 5 , 1982. 5 . Slot, J . W . & Geuze, H.J.: Sizing of protein A-colloidal gold probes for 6 . Wiley, L . M . & Calarca, P . G . : The effects of anti-embryo sera and their immunoelectron microscopy. J Cell Biol 90: 533-536, 1 9 8 1 . localization on the cell surface during mouse preiniplaritation development. Dev Biol 4 7 : 4 0 7 - 4 1 8 , 1 9 7 5 . Address for reprints: €3. Ove Nilssori Department of Human Anatomy Eiomedical Centre Box 5 7 1 S-751 23 Uppsala Sweden 153