Evaluation of a microplate latex agglutination method (Verotox-F assay) for detecting and characterizing verotoxins (Shiga toxins) in Escherichia coli

The performance of a commercial microplate latex agglutination assay, the V erotox-F assay, was compared with that of the Vero cell assay for the detec tion and characterization of Escherichia coil verocytotoxins (VTs). Culture filtrates of 68 VT-positive E. coil strains (65 human isolates [33 of sero type O157:H7/H-, 32 of non-O157 serotypes] and 3 reference strains) and 104 VT-negative strains (100 human isolates and 4 reference strains) were inve stigated. The toxin phenotypes and genotypes of the 68 VT-positive isolates were VT1 only (18 strains), VT2 and/or VT2c (33 strains), and VT1 plus VT2 (17 strains). The Verotox-F assay involved incubation of serial dilutions of culture filtrates with equal volumes of latex particles sensitized with anti-VT1 antibody or anti-VT2 antibody in 96-well microtiter plates with ap propriate controls and examination for latex agglutination after 20 to 24 h . Compared to the results of the Vero cell assay, the Verotox-F assay was 1 00% sensitive and 100% specific for the detection of VTs in culture filtrat es and correctly identified the toxin types of all 68 VT producers. By chec kerboard titration with purified toxins, the sensitivity of the Verotox-F a ssay was found to be 14 pg (0.7 ng/ml) for VT1, 12 pg (0.6 ng/ml) for VT2, and 350 pg (17.5 ng/ml) for VT2c; this sensitivity is comparable to that of the bioassay. The anti-VT2 latex reagent detected both Vn and VT2c and did not cross-react with VT1, The anti-VT1 reagent showed a low-level cross-re action with VT2c only at levels (greater than or equal to 4.5 mu g/ml) that were about 1,000-fold higher than those found in culture filtrates. We con clude that the Verotox-F assay is highly sensitive and specific for the det ection and characterization of VTs in culture filtrates of human E. coil is olates. The test is rapid, reliable, and easy to perform; its results are e asy to interpret; and it should allow testing for VT to become more widely performed.