A monoclonal antibody directed to N-acetylneuraminosyl-alpha 2 leads to 6-galactosyl residue in gangliosides and glycoproteins.

A hybridoma cell line producing a monoclonal antibody directed to N-acetylneuraminosyl-alpha 2 leads to 6-galactosyl residue has been established. The antibody is IgG2b and reacts only with lacto-series gangliosides as well as with glycoproteins having an N-acetylneuraminosyl-alpha 2 leads to 6-galactosyl residue, but does not react with gangliosides or glycoproteins having an N-acetylneuraminosyl-alpha 2 leads to 3- or -alpha 2 leads to 4-galactosyl residue. The antibody is useful for detecting the specific carbohydrate chain having this terminal structure by immunostaining of glycolipids separated on thin layer chromatography or glycoproteins separated on gel electrophoresis after blotting on nitrocellulose sheet. A remarkable accumulation of a few gangliosides having this terminal structure has been detected by this monoclonal antibody in some human cancer.

A hybridoma cell line producing a monoclonal antibody directed to N-acetylneuraminosyl-c2+6-galactosyl residue has been established. The antibody is IgG2b and reacts only with lacto-series gangliosides as well as with glycoproteins having an N-acetylneuraminosyl-c~2~6-galactosyl residue, but does not react with gangliosides or glycoproteins having an N-acetylneuraminosyl-c2+3-or -a2-4-galactosyl residue. The antibody is useful for detecting the specific carbohydrate chain having this terminal structure by immunostaining of glycolipids separated on thin layer chromatography or glycoproteins separated on gel electrophoresis after blotting on nitrocellulose sheet. A remarkable accumulation of a few gangliosides having this terminal structure has been detected by this monoclonal antibody in some human cancer.
Monoclonal antibodies directed to defined carbohydrate structures have been isolated and have proven to be useful probes in determination of the carbohydrate profile at the cell surface and expedient reagents in determination of carbohydrate structures (1)(2)(3)(4)(5)(6)(7)(8)(9). This approach has been conveniently applied for analysis of antigen profiles, in combination with the immunostaining of glycolipids separated on thin layer chromatography (10) and glycoproteins separated on gel electrophoresis followed by blotting on nitrocellulose sheet (11).
Recently, a new type of lacto-series ganglioside having a sialosyl-a2+6-galactosyl terminus was isolated and characterized from human erythrocytes (12) and human meconium (13). Two gangliosides having this terminal structure, sialosyllacto-neotetraosylceramide and sialosyl-lacto-norhexaosylceramide, were isolated and characterized as minor components of human erythrocytes (12). The former ganglioside was found to be the major component of human meconium (13). Glycoproteins having the carbohydrate chain with the terminal sialosyl-a2+6-galactosyl residue have been found to be widely distributed (see for a review Ref. 14), particuIarly in blood plasma glycoproteins (e.g. . The present paper describes establishment of a hybridoma secreting the IgG2b antibody, which reacts specifically with the sialosyl-a2-6-galactosyl structure in gangliosides as well as in glycoproteins, and the application of this antibody to detect this structure at nanogram order. ' "Materials and Methods" are presented in miniprint as prepared

RESULTS
Production of the Hybridoma-Fusion of the NS/1 myeloma with spleen cells of mice immunized against sialosyl-a2+6lacto-neotetraosylceramide yielded about 3% positive clones by the first assay after the fusion. Two clones, IB2 and IB9, were isolated which appeared to have the same reactivity, specifically to sialosyl-a2-6-lacto-neotetraosylceramide, but showed no reactivity to sialosyl-a2~3-lacto-neotetraosylceramide. They were derived from the same well (lIIC8) of the original fusion plate and were derivatives of the same clone. Both hybridomas, IB2 and IB9, can be propagated in BALB/ c mice as ascites form, producing high titer antibody. Antibodies from these hybridomas were identified as IgG2b class, and were successfully propagated as ascites form in "pristane"-treated BALB/c mice.
Specificity of the Antibody-Antibodies produced by both hybridomas, IB2 and IB9, showed identical specificity, and the supernatant fluid of the hybridomas showed the same specificity as ascites. The results described in this paper are those with the IB9 antibody. The reactivity of the antibody with various gangliosides and glycoproteins is shown in Figs. 1 and 2, respectively. It is clear that only lacto-series gangliosides containing the sialosyl-a2+6-galactosyl residue showed a specific reactivity. All other gangliosides failed to react with the antibodies present in the supernatant or in ascites. Human plasma fibronectin was shown to have an N-acetylneuraminosyl-cu2-+6-galactosyl residue (17), and gave a positive reaction with this antibody (Fig. 2 A ) . In contrast, the majority of the sialosyl residue in bovine plasma fibronectin is linked through the cu2-+4Gal structure and only a minority is linked through the n2+6Gal structure (25). This reflects a low reactivity of bovine fibronectin with this antibody (Fig. 2B). Bovine submaxillary mucin, which has the NeuAcaB-, 6GalNAc structure (14), showed a strong reactivity with this antibody (Fig. 2C). However, human erythrocyte glycophorin (27), which has a major oligosaccharide chain with the structure NeuAc~2--*3Gal/3l"t3(NeuAccu2+6)GalNAc (as), did not show any appreciable reaction with this antibody (Fig.  20), although glycophorin has one asparagine-linked complex-type oliogasaccharide containing the NeuAca24Gal residue as a minor component (29). The reactivity of human plasma fibronectin and bovine submaxillary mucin to this antibody was abolished by treatment with Vibrio cholerae sialidase (see Fig. 2, A and C). The weak reactivity of bovine plasma fibronectin to this antibody was slightly reduced by sialidase treatment. These results clearly indicate that the antibody recognizes NeuAcn2+6Gal and NeuAca2+ Dilution of Anttbody (ascites) 6GalNAc at the terminus. The latter structure at the internal residue, such as found in glycophorin (28), was not reactive.
Other types of sialosyl linkages in various glycoproteins, i.e. a2-3Ga1, a24Ga1, and cu24GlcNAc, were not reactive to this antibody. Determination of Gangliosides and Glycoprotein Profiles Having NeuAca24Gal Residue-Ganglio-series and lactoseries gangliosides having the NeuAca24Gal residue were separated on high performance thin layer chromatography and immunostained as shown in Fig. 3. None of the ganglioseries gangliosides were stained (Fig. 3, Lane 5 ) . A doublet corresponding to sialosyl-cu2+6-lacto-neotetraosylceramide (band h) and a band corresponding to a sialosyl-c~2+6-lactonorhexaosylceramide (band i) were stained (Lane 6). In addition, a slower migrating band was detected which has not been characterized (Lane 6). This component could be contamination present in the reference sample of G9' ganglioside (30). A ganglioside fraction isolated from two cases of human cancer (Fig. 3, Lanes 3 and 4 ) and their immunostaining pattern (Lanes 7 and 8) are also shown. GH3 was the major component in both cases (Lanes 3 and 4 ) ; however, a glycolipid showing a doublet corresponding to sialosyl-cu2-4-lacto-neotetraosylceramide (a doublet in Lane 3 corresponding to band h in Lane 2) was found as the second major component in one Bands a-fare, respectively, G M~, G M~, GM,, Gola GDlb, and GTlb gangliosides. Designation of these brain gangliosides is according to Svennerholm (33). Lane 2 is a mixture of standard lacto-series gangliosides isolated and previously characterized (12). A doublet band g represents sialosyl-a2+3-lacto-neotetraosylceramide (the two bands represent different ceramides); a doublet band h represents sialosyl-a2+6-lacto-neotetraosylceramide (the two bands represent different ceramides); band i, sialosyl-a2+6 lacto-norhexaosylceramide; band j , G9 ganglioside (a branched fucoganglioside with ceramide decasaccharide structure (30). Lane 3, ganglioside fraction of hepatocarcinoma (diagnosed as adenocarcinoma). Lane 4, ganglioside fraction of primary lung carcinoma metastatic to liver. FIG. 4 (right). Immunostaining of fibronectins separated on gel electrophoresis followed by blotting on nitrocellulose sheet. Lane 1, human plasma fibronectin; Lane 2, thermolysin digest of human plasma fibronectin (31); Lane 3, desialylated human plasma fibronectin by hydrolysis in 1% acetic acid. Left margin shows identification of the fragments. FN, intact fibronectin. (Lane 7). In addition, slower migrating bands, including the one corresponding to sialosyl-a2+6-lacto-norhexaosylceramide and another with slower mobility, were detected as being stained by the antibody (Lane 7). This type of large accumulation of gangliosides having the NeuAca2-6Gal residue was not observed in the other case of human cancer (lung cancer metastatic to liver) shown in Lanes 4 and 8. The major gangliosides chemically detectable in this case were GM:< and sialosyl-a2+3-lacto-neotetraosylceramide (Lune 4 ) , which were not immunostained by this antibody (Lune 8). Only a small quantity of sialosyl-a24-lacto-neotetraosylceramide was detected, which was weakly immunostained by this antibody (Lane 8).

case (Lane 3). This component was immunostained intensely by the antibody
As an example of glycoproteins having the NeuAca24Gal residue, human plasma fibronectin, its thermolysin digest, and desialylated products were separated on gel electrophoresis and immunostained after blotting on nitrocellulose sheet. An intense band was immunostained for intact fibronectin (Fig. 4, Lane I), and a doublet with M, = 140,000-150,000, 105,000,44,000, and 23,000 was immunostained after thermolysin digestion (Lune 2) (31). An intense fibronectin stain was completely lost after the sialosyl residue of fibronectin was eliminated (Lane 3).

DISCUSSION
Profiles of cell surface carbohydrates have been probed and defined by lectins (32). This approach has been strengthened and partially replaced by monoclonal antibodies directed to defined carbohydrate structures. Another potentially important use of anti-carbohydrate monoclonal antibodies is their application in structural analysis of carbohydrates. The im-Monoclonal Antibody to NeuAca24Gal munostaining procedure recently developed on thin layer chromatography (10) and blotting of electrophoretic gels on nitrocellulose sheets (11) is highly sensitive and requires only nanogram quantities of material. Immunostaining of complex carbohydrates by multiple monoclonals in combination with enzymatic hydrolysis will be potentially useful to elucidate the structure of nanogram quantities of carbohydrates, if the necessary number of well defined monclonal antibodies are available. Efforts to gain increasing numbers of well defined monoclonal anti-carbohydrate antibodies are essential to reach such a goal.
The antibody described in this paper defines the terminal residue NeuAca24Gal in glycolipids and glycoproteins and the NeuAca24GalNAc residue in glycoproteins. The antibody does not react with the internal sialosyla2+6GalNAc residue (28) linked to polypeptides since glycophorin, which contains this structure, was not reactive.
The structure NeuAca2+6Gal (or GalNAc) is known to be widely distributed in a variety of glycoproteins as the terminus of the short 0-linked oligosaccharide in mucin-type glycoproteins, i.e. NeuAca2+6GalNAcal+O-Ser (Thr), and as the terminus in the side chain of a complex type asparaginelinked structure, i.e. NeuAca2-+6Gal~l+4GlcNAc~l+ 2Manal-R (see for a review Ref. 14), although such a structure has been confirmed only relatively recently based on methylation analysis (15)(16)(17)25). Determination of such structures in glycoproteins requires isolation of oligosaccharides or glycopeptides after degradation, and methylation analysis before and after desialylation, and needs at least 0-200 pg of glycopeptides. If glycoproteins contain 3-5% carbohydrates (like fibronectin), a minimum quantity to obtain the information on sialosyl linkage would be at least 2-3 mg of glycoprotein. The results shown in Figs. 3 and 4 required only 100 ng of the glycoprotein. Since neither a ganlioside having N-glycolylneuraminosyl-a24-galactosyl residue nor a glycoprotein exclusively containing this structure have been isolated, we have had no chance to test the reactivity of the IB9 antibody to this structure.
Gangliosides with this structure have been isolated and characterized only recently (12,13). Two types of gangliosides have been isolated and characterized from human erythrocytes (12); one is sialosyl-a2+6-lacto-neotetraosylceramide (G4) and the other is sialosyl-a24-lacto-norhexaosylceramide (G7). The quantity of these gangliosides in human erythrocytes was very low (G4 and G7 comprised 1.9 and 1.4% of the total gangliosides of human erythrocytes, respectively) (12). Normal colonic mucosal tissue and normal liver contain similar quantities of these gangliosides. Interestingly, the quantity of gangliosides having this terminal structure was found to be much higher in some human cancers, and sialosyl-a2-6-lacto-neotetraosylceramide is a major ganglioside of some human cancers. The ganglioside composition of only two cases of human cancer are presented in this paper, as shown in Fig. 3. One accumulated sialosyl-a2+6 gangliosides and the other did not. However, we have observed many other cases, and the results of these studies will be published elsewhere. Since sialosyl-~2-+6-lacto-neotetraosylceramide is the major ganglioside in meconium (13), but represents only a minor component in various normal tissues, the remarkable accumulation of this ganglioside in human cancer may reflect an oncofetal expression of this ganglioside. The antibody is useful in determining the profile of gangliosides and sialosyl structures in tissues and cells.