Immunochemical Studies of Starfish Gangliosides: Production of Monoclonal Antibody against AG-2, the Major Ganglioside of Starfish Acanthaster planci, and Detecting Its Distribution in Tissues by TLC Immunostaining

  • Miyamoto, Tomofumi (Graduate School of Pharmaceutical Sciences, Kyushu University) ;
  • Yamamoto, Atsushi (Graduate School of Pharmaceutical Sciences, Kyushu University) ;
  • Sakai, Maki (Graduate School of Pharmaceutical Sciences, Kyushu University) ;
  • Tanaka, Hiroyuki (Graduate School of Pharmaceutical Sciences, Kyushu University) ;
  • Shoyama, Yukihiro (Graduate School of Pharmaceutical Sciences, Kyushu University) ;
  • Higuchi, Ryuichi (Graduate School of Pharmaceutical Sciences, Kyushu University)
  • Published : 2006.12.31

Abstract

In this study, we establish a thin-layer chromatography (TLC) immunostaining method for detecting starfish gangliosides. A new monoclonal antibody (MAb) against AG-2, the major gangliosides molecular species of Acanthaster planci, was produced by fusing hybridoma with splenocytes immunized to liposomal AG-2. BALB/c male mice were injected with liposomal AG-2 antigen, and immunized. Their splenocytos were isolated and fused with hypoxanthine-aminopterine-thimidine (HAT)-sensitive mouse myeloma cells. Hybridomas producing MAb reactive to AG-2 were cloned using the limited dilution method. Established hybridomas were cultured in eRDF medium. Crude MAb produced from clone 8D4 was purified with a magnesium pyrophosphate column. Enzyme immunoassay and TLC immunostaining of AG-2 were performed using the purified MAb. Structurally related gangliosides did not cross-react with anti-AG-2 antibodies. The detection limit of TLC immunostaining was 50 ng of AG-2. The newly established immunostaining method was further developed for detecting AG-2 distribution and qualitative analysis in tissues and/or organs. Our results show that the majority of AG-2 is present in the stomach of male A. planci, while AG-2 is distributed not only in the stomach but also in the the pyloric caeca of female A. planci.

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