Identification of Shiga toxins in Shiga toxin-producing Escherichia coli using immunoprecipitation and high-performance liquid chromatography-electrospray ionization mass spectrometry.
We developed a rapid and reliable identification method for Shiga toxins in Shiga toxin-producing Escherichia coli (STEC) using immunoprecipitation and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Polyclonal antisera specific for Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) were raised in rabbits so as to be used for the immunoprecipitation. The immunoprecipitaion was carried out by mixing sample solutions with 50 microl each of the antisera to Stx1 and Stx2 followed by allowing the mixed solutions to stand for 30 min. The quantity required to obtain the immunoprecipitate was more than 0.5 microg of Shiga toxins. HPLC-ESI-MS analysis of the resulting immunoprecipitates provided accurate molecular weight information on Shiga toxins, leading to direct evidence for the presence of these toxins. It requires at most two days to perform our procedure from toxin extraction to measurement of HPLC-ESI-MS whereas the previous method using isolation procedures required about two weeks to complete. The usefulness of the present method has been demonstrated by identifying Stx1, Stx2 and a variant of Stx2 (Stx2e) in the immunoprecipitates prepared from STEC strains.
Kondo
Fumio
F
Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan. fumio@ipc-tokai.or.jp
Saito
Hiroshi
H
Hayashi
Rumiko
R
Onda
Hiroyuki
H
Kobayashi
Shinichi
S
Matsumoto
Masakado
M
Suzuki
Masahiro
M
Ito
Yuko
Y
Oka
Hisao
H
Nakanishi
Toyofumi
T
Shimizu
Akira
A
D004926:Escherichia coli / Q000737:chemistry*; D011233:Precipitin Tests / Q000379:methods; D022603:Shiga Toxins / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D022603:Shiga Toxins