An improved immunochromatographic test strip that sensitively detects heat-labile enterotoxin produced by enterotoxigenic Escherichia coli.

Abstract

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.