Arimitsu Hideyuki

Enterotoxigenic Escherichia coli (ETEC) can cause watery diarrhea not only in humans but also in domestic animals, for example, causing post-weaning diarrhea in piglets. Because the major causative factors of ETEC are heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), rapid detection methods are required for these toxins. We previously reported a prototype immunochromatographic (IC) test strip for LT comprising a mouse monoclonal antibody (mAb) and rabbit polyclonal antibody. However, the toxin detection limit of this test strip was insufficient, and the test sample bacteria needed to be cultured overnight with lincomycin supplementation to enhance LT production. Moreover, no IC test strips were available for ST. Therefore, we attempted to create a chimera protein of the B subunit of LT (LTB) and ST and develop mAbs against both LTB and ST through immunization of mice. Although nine antigen-specific mAb clones were obtained, all were LTB-specific. IC test strips prepared with the mAb 31D11 and mAb 34D4 pair were able to detect 0.15 ng/150 μL of purified LT. In addition, this IC test strip was able to detect LT in 6-h culture supernatants of clinical isolates from swine and human without requiring lincomycin supplementation. Quantification of LT levels using sandwich ELISA corroborated the IC results, indicating that the improved IC test strip enabled the detection of LT at lower concentrations and with shorter culture times than the previous method, without the need for supplements. This test strip will be useful for ETEC detection in the food hygiene and livestock hygiene fields.

Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.