In vitro immunization of rat spleen lymphocytes with Forssman glycosphingolipid antigen and the generation of a monoclonal antibody

J. K. Gathuru, I. Miyoshi, M. Naiki

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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-μ was used to support B cell differentiation. Unprimed rat spleen cells (2 × 106/ml) were stimulated with 2 μg/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 (GalNAcα1-3GalNAcβ1-3Galα1-4Galβ1-4Glc-ceramide).

Original languageEnglish
Pages (from-to)95-102
Number of pages8
JournalJournal of Immunological Methods
Volume137
Issue number1
DOIs
Publication statusPublished - 1991
Externally publishedYes

Keywords

  • Forssman glycosphingolipid
  • In vitro immunization
  • Monoclonal antibody
  • Tumor-associated antigen

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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