The Journal of Cell Biology, Vol 93, 591-602, Copyright © 1982 by The Rockefeller University Press
Immunofluorescent detection of erythrocyte sialoglycoprotein antigens on murine erythroid cells
AH Sarris and GE Palade
A sialoglycoprotein fraction isolated from murine (DBA/2) erythrocytic
ghosts (see companion article, Sarris and Palade, 1982, J. Cell. Biol.
93:583-590) was used to raise antibodies in rabbits. By immune-IgG
(serum)-[125I] protein A overlays, the antibodies were found to react
positively with the four 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
polyacrylamide gel electrophoretograms of solubilized ghosts, and with a
minor sialoglycoprotein hidden under this trail. IgG's 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 three 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 sack), and
in the 14-d 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 IgGs gave negative reactions in
all cases. The antibodies were species-specific: they did not crossreact
with either human or rat erythrocytes.
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