Red Cell Membrane Antigens Common to Several Mammalian Species 1

Abstract

Differences in protein composition of red cell membranes of non-human primates and red cell membranes of several mammalian species were demonstrated by physicochemical methods (1, 2). Immunological studies of solubilized human red cell membrane proteins have detected 5-7 antigenic determinants associated with such protein components. The majority of these antigens were shown to be localized in deeper layers of the membrane (3). Studies on one of the membrane proteins (spectrin) isolated from human and three other mammalian red cell membranes have shown that they are immunologically related (4). This preliminary report describes our studies on the immunologic reactivity of red cell membrane proteins of several mammalian species with antisera prepared against human membrane proteins.

Materials and Methods. Erythrocytes. Human red blood cells were collected in acid-citrate—dextrose and stored no longer than two to three weeks prior to use. Sheep, ox, rat, pig, horse, cat, dog, and rabbit red cells were obained in Alsever's solution from Pentex Biochemicals (Miles Laboratories, Kankakee, IL). The cells were washed four times with isotonic saline before hemolysis.

Preparation of Stromal Proteins. Ghosts were prepared by osmotic lysis and phosphate buffer washes according to the method of Rega et al. (5) until complete removal of hemoglobin. Stromata were extracted in the cold with n-butanol at pH 2.1 (6). The waterphases thus obtained were dialyzed against distilled water and concentrated by pervaporation prior to gel filtration.

Solubilization of stromal proteins was also performed by dialysis of hemoglobin free ghosts against water alkalinized to pH 9.5 (7).

Chromatography. The waterphases were chromatographed on Bio-gel P-100 (Bio-Rad Laboratories Calbiochem, Los Angeles, CA) in formic acid (0.05 M) as previously described (4, 6).

Immunoelectrophoresis and immunodiffusion. The micro method of Scheidegger was performed in 1% agar in barbital, calcium lactate buffer (8).