In order to detect immunoglobulin (Ig)A and IgG antibodies to Escherichia coli-secreted protein B in sera of children infected with Shiga toxin-producing Escherichia coli, an enzyme-linked immunosorbent assay was developed. The assay was tested using acute sera from 40 children with diarrhea-associated hemolytic uremic syndrome compared with 238 sera obtained from pediatric controls. Two cut-off values were used for children <5 (n=27) or > or =5 (n=13) years of age. Among the younger patients, 24 of 27 had IgA antibodies to Escherichia coli-secreted protein B (sensitivity, 89%; specificity, 98%) and 22 of 27 had IgG antibodies (sensitivity, 82%; specificity, 94%). Among the older patients, 13 of 13 had IgA antibodies (sensitivity, 100%; specificity, 96%) and 11 of 13 had IgG antibodies (sensitivity, 85%; specificity, 96%). This enzyme-linked immunosorbent assay detects Shiga-toxin-producing Escherichia coli independent of serogroup and could serve as a complementary assay for detection of infection.