Glycolipid-directed FH6 monoclonal antibody recognizes high molecular weight glycoprotein antigen carrying sialyl Lex-i determinant in the culture supernatant of PC-9 cells.
The properties of the antigen recognized by monoclonal antibody FH6 have been analyzed. FH6 was originally generated against a glycolipid, i.e. a difucoganglioside isolated from human colonic adenocarcinoma, and specifically reacts with sialyl Lex-i determinant. Several culture supernatants of human carcinoma cell line cells were found to have high levels of FH6-reactive antigen, and PC-9, a human lung carcinoma cell line was used for the analysis. A solid-phase sandwich radioimmunoassay was performed to detect the antigen. The antigenic activity was extractable in 0.6 M PCA or 7% TCA, and was sensitive to mild alkaline treatment and to Pronase digestion. Most of the antigen was eluted in the void volume of a Sepharose CL-2B column, which indicates that its molecular weight is greater than several million. It was eluted from a DEAE-cellulose column at a NaCl concentration in the range of 0.2-0.25 M. The immunoaffinity-purified antigen has a high carbohydrate content of more than 80%. These data indicate that the antigen recognized by FH6 in the culture supernatant of PC-9 is not a glycolipid, but a high molecular weight glycoprotein which could be referred to as a mucin, or a proteoglycan, which contains keratan-sulfate like glycosaminoglycan chains, as judged from the results of the glycosidase treatments.
Yamagata
Y
Y
Department of Clinical Science and Laboratory Medicine, Kyoto University Faculty of Medicine, Japan.
Shigeta
K
K
Masuda
T
T
Imura
H
H
Murachi
T
T
Kannagi
R
R
Shin
S
S
Tachikawa
T
T
Noda
A
A
Ohe
Y
Y
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000276:immunology / Q000302:isolation & purification*; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D006017:Glycolipids / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D006026:Glycoside Hydrolases; D006801:Humans; D016256:Lewis X Antigen; D008175:Lung Neoplasms / Q000276:immunology; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D003470:Culture Media; D006017:Glycolipids; D006023:Glycoproteins; D016256:Lewis X Antigen; D006026:Glycoside Hydrolases