Terminal Β-galactose Research Articles

Characterization of the antibodies in normal rabbit serum responsible for the lysis of NK-sensitive target cells

Normal rabbit serum has previously been reported to lyse a spectrum of murine lymphoid cell lines characterized by being susceptible to natural killer (NK) cells. Although we have failed to confirm that a simple correlation exists between the susceptibility of target cells to NK cells and to normal rabbit serum, we were able to characterize a number of naturally occurring anti-carbohydrate antibodies which selectively lyse NK-susceptible lymphoma cells. The antibodies present in normal rabbit serum responsible for lysing an NK susceptible clone derived from the DBA/2 lymphoma L5178Y (27v−IC2) were found to be directed against the terminal β- N-acetylgalactosamine sugar present on the neutral glycolipid gangliotriosylceramide. Interestingly, normal rabbit serum did not lyse an NK-insusceptible variant, derived from the same lymphoma, which lacked gangliotriosylceramide. YAC-1 target cells, the prototype target for murine NK cells, were insusceptible to normal rabbit serum. However, following neuraminidase treatment, such cells were lysed by an antibody in normal serum directed against terminal β-galactose residues. The anti-β-galactose reactivity was directed against the carbohydrate structure of gangliotetraosylceramide. These findings are discussed in light of the hypothesis that NK cells interact with susceptible target cells through cell surface glycoconjugates.

Immune reactivities of antibodies against glycolipids—II comparative properties, using liposomes, of purified antibodies against mono-, di- and thihexosyl ceramide haptens

Specific antibodies against ceramide monohexoside (CMH), ceramide dihexoside (CDH) and ceramide trihexoside (CTH) were purified from antiserum or normal (unimmunized) rabbit serum by using liposomes as immunoadsorbents. Purified antibodies were compared with whole antiserum, or immunologically reactive normal rabbit serum, for specific activity against the immunizing antigen, and cross-reactivity with the other haptens. High levels of reactivity and reciprocal cross-reactivity were observed both with antisera and purified antibodies against CMH and CDH. In contrast, although anti-CMH and anti-CDH sera both apparently ‘cross-reacted’ with CTH, the purified antibodies in each case did not have any detectable cross-reactivity with CTH. Conversely, anti-CTH antiserum apparently ‘cross-reacted’ with CDH, whereas purified antibodies did not. It was concluded that rabbit antiserum sometimes may contain naturally-occuring antibodies against a glycosphingolipid, and these may be eliminated by using purifief non-cross-reacting antibodies. Naturally-occurring antibodies against CHD and CTH, but not against CMH were found in 10 normal rabbit sera. Purified naturally-occurring anti-CDH antibodies obtained from one of these rabbits did not cross-react with CTH, but purified anti-CTH from the same rabbit did cross-react with CDH. Pools of ‘high activity’ or ‘low activity’ anti-CMH antibodies were obtained from a single rabbit by using antisera taken at different times in the immunization schedule. These antibodies were tested for activity against CMH and CDH (both having a terminal β-galactose), and CTH and digalactosyl diglyceride (both having a terminal α-galactose). Cross-reactivity, as above, was not observed with CTH. High activity anti-CMH antibodies, however, reacted equally well with CDH and digalactosyl diglyceride. Based on this it was concluded that anomeric configuraton alone did not necessarily determine specificity of purified antibodies. In contrast, the low antibodiies, in quantities which gave the same high degree of reactivity with the immunizing antigen observed with high activity antibodies, showed only very low levels of cross-reactivity with either CDH or digalactosyl diglyceride. It was concluded further, therefore, the cross-reactivity may depend to some extent on relative antibody activity (which may be related to antibody affinity) against the immunizing antigen.