In vitro immunization of rat spleen lymphocytes with Forssman glycosphingolipid antigen and the generation of a monoclonal antibody.
A procedure for in vitro immunization of splenic lymphocytes with a glycolipid antigen is described. Culture medium supernatant of ConA- and PHA-stimulated spleen cells and that of Con A-stimulated human Jurkat T cell line (IL-2-rich medium) were used as sources of cytokines to support T and B cell stimulation, and anti-mu was used to support B cell differentiation. Unprimed rat spleen cells (2 x 10(6)/ml) were stimulated with 2 micrograms/ml Forssman glycolipid antigen coupled to Sepharose for 4 days. The cells were fused with a mouse myeloma cell line P3-X63-Ag8-U1. At initial screening, 12% of the colony forming wells were secreting specific antibody. After cloning, a stable hybridoma cell line (designated 4C3) was established which secreted a monoclonal IgM antibody directed against the carbohydrate moiety of Forssman glycosphingolipid (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide).
Gathuru
J K
JK
Department of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Miyoshi
I
I
Naiki
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D005577:Forssman Antigen / Q000276:immunology*; D005915:Globosides / Q000276:immunology*; D007114:Immunization; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology*; D008297:Male; D051381:Rats; D013154:Spleen / Q000276:immunology*; D013997:Time Factors
D000911:Antibodies, Monoclonal; D005915:Globosides; C010163:Forssman glycolipid; D005577:Forssman Antigen