Rapid simultaneous detection of verotoxin 1 and 2 using time-resolved fluoroimmunoassay

• Published in: BUNSEKI KAGAKU Vol. 51; no. 6; pp. 381 - 387
• Main Authors: WATANABE, Kazuyuki, ARAKAWA, Hidetoshi, MAEDA, Masako
• Format: Journal Article
• Language: English; Japanese
• Published: Tokyo The Japan Society for Analytical Chemistry 01.06.2002; Japan Science and Technology Agency
• Subjects: enterohaemorrhagic Escherichia coli (EHEC); simultaneous measurement; time-resolved fluoroimmunoassay (TRFIA); verotoxin
• ISSN: 0525-1931
• DOI: 10.2116/bunsekikagaku.51.381

The infection of Enterohaemorrhagic Escherichia coli (EHEC), especially serotype O157 : H7, causes hemorrhagic colitis, and has been associated with hemolytic uremic syndrome or encephalopathy, which may lead to death. Consequently, the prevention of serious illness due to infection via early stage diagnosis is of the utmost importance. To this end, the development of detection methods that are simple, rapid and highly sensitive is necessary for EHEC. O157 produces two vero toxins, VT1 and VT2. VT1 is identical to the Shiga toxin, while the other type, VT2, is different from VT1. The conventional diagnosis of O157 is conducted after isolating bacteria from clinical specimens, followed by a serological determination and the identification of VTs. This method is both complicated and time-consuming. Recently, rapid, direct immunological methods for the identification of O157, i.e., immunochromatography and latex agglutination, have been developed. However, these techniques can not simultaneously measure these two toxins. On the other hand, the use of fluorescence lanthanide ions as a labeling material of antibodies and antigens makes it is possible to develop an immunoassay that can measure two or more subjects simultaneously. After excitation by short-pulse light, a fluorescence lanthanide ion, such as europium and terbium, exhibits a large Stokes shift and very narrow emission spectra that has no overlap with other lanthanide's emission spectra. In addition, their long fluorescence lifetimes allow the use of time-resolved fluorescence detection, which has led to the development of ultra sensitive time-resolved fluorometry (TRF). In this paper, we describe a sensitive and specific method based on time-resolved fluoroimmunoassay (TRFIA) for the simultaneous determinations of two verotoxins.