Different binding properties of three monoclonal antibodies to sialyl Le(x) glycolipids in a gastric cancer cell line and normal stomach tissue.
We established a mouse monoclonal antibody (Mab) KM93 which recognized sialyl Le(x)-carbohydrate epitope determined by solid phase radioimmunoassay using a panel of authentic glycolipids. The specificity of KM93 was similar to another anti-sialyl Le(x) Mab CSLEX-1 established previously, and different from that of Mab FH6 which recognized sialyl Le(x)-i (sialyl dimeric Le(x)). In a further study, however, we found that KM93 reacted with some glycolipids much more strongly than CSLEX-1 did on thin-layer chromatography (TLC) plates. We purified two gangliosides named K-1 and K-2 from gastric cancer cell line KATOIII, and three gangliosides named H-1, H-2, and H-3 from human stomach. KM93 reacted with all of these glycolipids. CSLEX-1 reacted with K-1 and K-2 with less intensity than KM93 did, and faintly reacted with H-1, but not at all with H-2 or H-3. FH6 did not react with K-1, K-2 or H-1, while it stained H-2 and H-3. In spite of this different reactivity with Mabs, analysis by proton nuclear magnetic resonance (1H NMR) proved that carbohydrate structure of K-1 and H-2 were the same: NeuAc alpha 2-->3Ga1 beta 1-->4 [Fuc alpha 1-->3] G1cNAc beta 1-->3 Ga1 beta 1-->4G1c beta 1-->1Cer. The H1 NMR spectrum of H-3 also indicated that H-3 consisted of the same sugars as K-1. These results indicated that KM93 had wider reactivity than CSLEX-1, and that the distinct reactivity of KM93 from CSLEX-1 was not caused by sugar moiety. It was also shown that the interaction of sialyl Le(x) sugar determinant with MAb depended on the tissue origin of molecules carrying carbohydrate.
Dohi
T
T
Division of Clinical Biochemistry, National Medical Center, Tokyo, Japan.
Nemoto
T
T
Ohta
S
S
Shitara
K
K
Hanai
N
N
Nudelman
E
E
Hakomori
S
S
Oshima
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005732:Gangliosides / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D013274:Stomach Neoplasms / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D005732:Gangliosides