Production of monoclonal antibodies specific for two distinct steric portions of the glycolipid ganglio-N-triosylceramide (asialo GM2).

Abstract

Two hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of BALB/c mice that had been immunized with the glycolipid ganglio-N-triosylceramide (asialo GM2). The specificity of the monoclonal antibodies produced by these hybridomas, one an IgM and the other an IgG3, has been defined by hemagglutination inhibition, complement fixation, and lysis of glycolipid liposomes by antibody and complement. A major determinant recognized by the IgM antibody is the nonreducing terminal N-acetylgalactosamine including the C6 primary hydroxyl group, but excluding the C2-acetamide group of N-acetylgalactosamine, because oxidation with galactose oxidase produced a structure showing only minimal cross-reaction with the IgM but replacement of the N-acetyl group with an N-n-butyryl group produced a glycolipid that reacts with IgM antibody to the same extent as with the unmodified glycoplipd. A major determinant recognized by the IgG3 antibody is the terminal N-acetylgalactosamine including the C2-acetamido group, but excluding the C6 primary hydroxyl group of N-acetylgalactosamine, because replacement of the N-acetyl group with an N-n-butyryl group produced a glycolipid that did not react with the IgG3 antibody; in striking contrast the IgG3 antibody reacted with the C6-oxidized glycolipid as well as with the native glycolipid. Neither antibody reacted significantly with any other natural glycolipids tested including several that are structurally related to asialo GM2 such as ganglioside GM2, ganglio-N-tetraosylceramide (asialo GM1), or ceramide dihexoside. These results indicated that in addition to the fine structure specificity described above both antibodies recognize the nonreducing terminal GalNAc beta 1 leads to 4Gal structure. The strict antigenic specificity of these monoclonal anti-glycolipid antibodies indicates their great potential as specific probes for cell surface studies.

Selective depletion of nk cell activity in vivo and its effect on the growth of NK‐sensitive and nk‐resistant tumor cell variants

“…The general usefulness of anti-interferon antibody as an NK-suppressive reagent is, however, compromised by two problems: (1) tumor cell replication is often directly inhibited by interferon derived from the same species (Gresser, 1977) and hence any promoting effect of anti-interferon antibody on the growth of syngeneic tumors could not be unambiguously assigned to its effects on the NK system; (2) such a reagent would not be useful in, for example, studying the role of NK cells in viral infections, where interferon is already established as a major component of host defenses. In keeping with these observatlons~ the rate Recently, we (Young et al, 1979) and others (Kasai of mice inoculated with 106 cl27v-lC2 tumor cells was et al, 1980), demonstrated that rabbit anti-asialosignificantly prolonged by poly-I:C injection (Fig. 4).…”

Generation and characterization of anti-sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve

“…In brief, 7-week-old female mice of the C3W HeN inbred strain (purchased from Japan Clea, Tokyo, Japan) were immunized with purified SGPG (100 pg, total amount per mouse) adsorbed to acid-treated Salmonella minnesota (250 pg total amount per mouse; Salmonella minnesota mutant R595 was kindly provided by Dr. T. Tomita, Institute of Medical Science, University of Tokyo) by the procedure of Young et al (1979). Each mouse received intravenous injections of the antigenbacteria complex on days 0 , 4 , 7, 11, and 21.…”

NK cell-target interactions: approaches towards definition of recognition structures

“…5 x 103 target cells were used per well (c) Aliquots of cells were tested for the presence of membrane associated Gg3cer by immunofluorescence using monoclonal anti-Gg3cer (IgM) and fluorescinated Goat anti-mouse immunoglobulins (d) The amount of Ggacer associated with c127v-1 C2 cells was estimated from the actual chemical amount of this glycolipid isolated from these cells. Gg3cer associated with 27av cells was determined in parallel experiments using 3H-labeled Gg3cer as a tracer (19). It was used at a final concentration of 1:1000.…”