DETECTION OF ESCHERICHIA-COLI STRAINS PRODUCING CYTOTOXIC NECROTIZINGFACTOR TYPE-2 (CNF2) BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Sheep and rabbit antisera were produced against lysates of E. coli str ain 711(pVir). This K-12 strain carries the Vir plasmid which codes fo r Cytotoxic Necrotizing Factor type 2 (CNF2). Immunoglobulin G (IgG) f ractions of both immune sera were subsequently purified by a two-step precipitation method. To increase the specificity for CNF2, the sheep Ige preparation was extensively adsorbed against both a sonicated extr act of isogenic K-12 strain 711 and intact phenol-treated cells of vac cine strain 711(pVir). An enzyme-linked immunosorbent assay (ELISA) wa s developed to detect clinical isolates of E. coli producing CNF2, usi ng the final preparations of rabbit and sheep IgG in a double sandwich technique. The results obtained with this CNF2-ELISA were compared to those obtained with the conventional HeLa cell cytotoxicity assay. Th e testing of 133 E. call strains (49 CNF2 positive strains and 84 nega tive strains) resulted in no false-negative and no false-positive. The refore, the CNF2-ELISA offers a good alternative to the HeLa cell cult ure assay for the detection of CNF2-producing strains where facilities for and experience with cell cultures is lacking.