Immunofluorescent detection of erythrocyte sialoglycoprotein antigens on murine erythroid cells.

Abstract

A sialoglycoprotein fraction isolated from murine (DBA/2) erythrocytic ghosts was used to raise antibodies in rabbits. By immune-IgG (serum)-[125I] protein A overlays, the antibodies were found to react positively with the 4 sialoglycoprotein monomers (gp-2.1, gp-2.2, gp-3.1 and gp-3.2) of the original fraction, with the sialoglycoproteins detected in erythrocytic ghosts (gp-2.1 and gp-3.1), with a diffuse component (probably a macroglycolipid) trailing around gp-3.1 in SDS [sodium dodecyl sulfate] polyacrylamide gel electrophoretograms of solubilized ghosts, and with a minor sialoglycoprotein hidden under this trial. IgG isolated from immune and nonimmune rabbit sera were conjugated to tetramethylrhodamine isothiocyanate and used to survey, by fluorescence microscopy, the distribution of the cognate antigens on the 3 different erythroid lines known to succeed each other during the life span of the mouse. In the peripheral blood of the adult, the antibodies recognized only mature erythrocytes; they did not crossreact with either platelets, monocytes or different types of granulocytes. In the spleen of adult anemic mice, the antibodies reacted weakly with proerythroblasts and strongly with all types of erythroblasts. In enucleating erythroblasts, antigens were preferentially segregated on the cell membrane of the nascent reticulocyte. In the 10 day old embryo, antigens were already present on the primitive nucleated erythrocytes (produced by the blood islets of the yolk sac) and in the 14-day fetus they were found on all hepatic erythroblasts and derived non-nucleated erythrocytes. A positive immunoreaction was also obtained on Friend erythroleukemic cells, before or after induction by dimethyl sulfoxide. Nonimmune serum, or nonimmune IgG gave negative reactions in all cases. The antibodies were species-specific: they did not crossreact with either human or rat erythrocytes.