Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin-producing Escherichia coli strains by a time-resolved fluorescence immunoassay.

A rapid and sensitive two-step time-resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin-producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu ) chelate was then used as a detector, followed by fluorescence measurements using time-resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1-1000 ng/ml). The intra- and inter-assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2-specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2-specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation-based platform and aid in the accurate and prompt diagnosis of STEC infections.