K. E. Caldwell

Immunochemical studies of infectious mononucleosis. IX. Heterophile antigen associated with a glycoprotein from the bovine erythrocyte membrane

A glycoprotein was isolated from the membrane of the bovine erythrocyte by refluxing the acetone‐ and ethanol‐extracted stroma residue with 75% ethanol. The glycoprotein was purified by phosphocellulose chromatography, ethanol precipitation, lipid‐solvent extraction and DEAE chromatography. The glycoprotein appeared to have two serological determinants, both reactive with antibodies present in the sera of patients with infectious mononucleosis. One of the determinants is similar to the Paul‐Bunnell heterophile antigen found on sheep erythrocytes. It is dependent on carbohydrate, including sialic acid residues. Another specificity, seemingly not shared by sheep erythrocytes to any great extent, is resistance to neuraminidase and to alkaline borohydride treatment and thus it may be located either on the polypeptide portion of the molecule or on an alkali‐stable oligosaccharide. The purified glycoprotein comprises 73% amino acids. Carbohydrate components and their molar ratios were sialic acid (1.0): galactose (1.5): N‐acetylglucosamine (1.1): N‐acetylgalactosamine (0.5): mannose (0.1).

Purified membrane fractions from mammary tumor cells elicit biological reactivity in in vitro cell-mediated immune reactions

Lithium chloride (LiCl) aggregation followed by centrifugation through a sucrose step gradient was used to obtain purified plasma membranes from two sublines of mouse mammary adenocarcinoma. These two tumor lines were chemically induced by treatment of a hyperplastic nodule with 7,12-dimethylbenzanthracene (DMBA). One line, D1-DMBA-3, has been found to be immunogenic to the host of origin, while the other, D1-DMBA-2, does not elicit specific tumor immunity as previously tested in in vivo and in vitro immune reactions. The purified membrane fractions were assayed for protein, DNA and sialic acid content as well as enzymatic markers of membrane purity. When tested in blastogenesis and cytotoxicity reactions, membrane-containing fractions from the D1-DMBA-3 immunogenic tumor were found to be stimulatory to spleen cells of D1-DMBA-3 tumor bearers over a ten-fold protein concentration. Spleen cells from normal mice do not respond in these reactions to the various tumor fractions. No reactivity was observed when non-membrane-containing preparations were used as stimuli in the cell-mediated immune reactions. The specificity of these reactivities was further demonstrated by the lack of responses when fractions from the non-immunogenic D1-DMBA-2 tumor were tested in parallel in our in vitro assays. The data presented indicate that the procedure employed is useful for the isolation of membrane-associated, tumor-specific antigens which can be easily quantitated and still retain biological activity in in vitro tests of cell-mediated immunity.

Immunochemical studies of infectious mononucleosis—X. Characterization of a glycoprotein from horse erythrocytes which reacts with Paul-Bunnell antibody☆

A highly purified preparation of horse erythrocyte glycoprotein was prepared from an aqueous ethanolic extract of hemoglobin-free membranes. The subunit apparent mol. wt was 30,000. In aqueous solution the glycoprotein formed globular aggregates of 93 +/- 16 A diameter. The glycoprotein had a receptor for the Paul-Bunnell antibody of infectious mononucleosis which was associated with an O-glycosidically linked oligosaccharide and dependent on the presence of N-glycolylneuraminic acid. In addition the glycoprotein had a neuraminidase-sensitive receptor for human peripheral blood lymphocytes. Fifty per cent inhibition of the rosetting of sheep red cells by 4 x 10(5) lymphocytes was caused by 30 microgram of glycoprotein.