Purpose: To generate monoclonal antibodies against intimin-γ (intG) and intimin-α (intA) for detecting EHEC.
Methods: The gene encoding the N-terminal variable region of the intG from E. coli O157:H7 and intA of IEH EPEC strain 8111 were PCR-amplified, cloned and expressed. The gene products were purified and the recombinant intG and intA were used to prepare monoclonal antibodies (mAb) which were characterized using ELISA, Western blot and immunofluorescent microscopy. The anti-intA was used to prepare immunomagnetic particles (IMP).
Results: The recombinant N-terminal region of intG and intA were used to generate anti-intG 5F11 and anti-intA 20C6 mAb. mAb 5F11 recognized the recombinant intG and the 97-KDa protein from E. coli O157:H7 lysate, while mAb 20C6 recognized only those of the EPEC strain 8111. Immunofluorescent microscopy using FITC-labeled mAb suggested that 5F11 and 20C6 attached to the surface of E. coli O157:H7 and EPEC strain 8111, respectively. mAb 20C6-IMP was prepared, and used to concentrate EPEC strain 8111 grown in Dulbecco’s modified Eagle’s medium (DMEM). Approximately 20% of the cells were recovered after treatment with 20C6-IMP.
Significance: The monoclonal antibodies recognized both the recombinant and native intimins from cell lysates of either E. coli O157:H7 or EPEC strain 8111, and could be used for detecting these isolates. The anti intimin mAb can be attached to IMP and used to concentrate intimin-producing EHEC and EPEC.