Shiga-like toxin II (SLT-II) was purified to apparent homogeneity from Escherichia coil K-12 strain NM522 containing the cloned toxin genes on recombinant plasmid pEB1. Purification was accomplished by a series of column chromatography techniques: anion-exchange, chromatofocusing, cation-exchange, and monoclonal antibody affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the pure toxin showed that SLT-II consisted of A and B subunits with apparent molecular weights of 32,000 and 10,200 + 800, respectively. A band of molecular weight 25,000 was also observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as the Al subunit by Western immunoblot analysis with toxin-specific monoclonal antibodies (MAbs). The pl of the purified toxin was 5.2. Approximately 1 pg of pure SLT-ll was a 50% cytotoxic dose for HeLa cells. The toxin was neutralized by polyclonal antibodies and MAbs to SLT-II, but was not neutralized by polyclonal antibodies or MAbs to SLT-I. Five hybridomas against SLT-II were produced (BC5 BB12, DC1 EH5, EA5 BA3, ED5 DF3, and GB6 BA4). Culture supernatant fluids containing MAbs from these hybridomas did not neutralize the cytotoxicity of SLT-I or Shiga toxin. Western blot analysis showed that two MAbs (MAb DC1 EH5 and MAb GB6 BA4) recognized the A and Al subunits of SLT-I and three MAbs (MAb BC5 BB12, MAb EA5 BA3, and MAb ED5 DF3) recognized the B subunit of SLT-II. MAb BC5 BB12 was used to prepare an affinity column for toxin purification.

Shiga-like toxin (SLT)-producing Escherichia coli has been associated with a spectrum of human illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. It produces at least two antigenically distinct toxins designated SLT-I and SLT-II, which have been implicated in disease. Currently available toxin assays, however, are not suitable for most clinical or public health laboratories. In this study, we have developed two sandwich enzyme-linked immunosorbent assays (ELISAs) based on toxin-specific murine monoclonal capture antibodies and rabbit polyclonal second antibodies which are specific for SLT-I and SLT-II. The SLT-I ELISA detected 200 pg of purified SLT-I, and the SLT-II ELISA detected 75 pg of purified SLT-II. The types of SLT produced by 166 human and 54 animal isolates of E. coli that produced moderate to high levels of toxin were determined by the ELISA, and results were confirmed by cytotoxin neutralization assays. With the exception of results from three strains, the tests agreed on the types of toxin present. DNA probe assays of 86 of 87 isolates also agreed with the ELISA and neutralization results. Although the SLT-II ELISA was specific for the SLT-II variant produced by porcine edema strains, most of the isolates examined produced levels of toxin (<50 50%