小麦Lipid Transfer Proteinに対する酵素免疫測定法の確立

Abstract

Human serum showing high IgE contents to wheat reacted with about 9kDa protein in crude extract of wheat flour on Western analysis. The 9kDa protein was purified from the extract by ammonium sulfate precipitation and anion-exchange, cation-exchange, and hydrophobic chromatography successively. The Nterminal amino acid sequence of this protein was determined to be homologous to non-specific Lipid Transfer Protein (LTP). Sandwich enzyme-linked immunosorbent assay (ELISA) was constructed with rabbit polyclonal IgG raised against this protein. The detection limit of this ELISA was 5 ng/ml for wheat LTP and the cross-reactivity to barley LTP was negligible. Since 2002 in Japan, commercial ELISAs for gliadin were noticed ministerialy to use for the determination of the content of wheat proteins in foods. It is difficult for the noticed ELISA to be applied to fermented foods because of proteolysis of gliadin. However, LTP could be determined in soy sause and wheat-supplemented beer by our ELISA for LTP. It was shown that LTP is an appropriate target protein for the determination of ingredient contents even in fermented foods because of its proteolysis-resistant nature.