Development Of Monoclonal Antibodies Against The LPS Of Entero-hemorrhagic E. Coli O157and Preparation Of The Immunomagnetic Beads Coated With The Monoclonal Antibody

Shiga toxin-producing E. coli (STEC), also named Vero toxin-producing E. coli (VTEC), is an important pathogen in human. It has been known that more than100different serotypes of E. coli can produce Shiga toxin, including the O157:H7serotype, which has high pathogenicity. Generally, E. coli isolated from infected humans or domestic animals, having the plasmid pO157and able to cause attaching and effacing lesions, are collectively referred to as entero-hemorrhagic E. coli (EHEC).Infectious diarrhea caused by EHEC is one of the most important public health problems in the world. The EHEC O157:H7, which is highly pathogenic to humans and animals, is the representative causative agent. Therefore, it’s necessary to detect and monitor the disease rapidly and efficiently.In the study, six monoclonal antibodies (MAbs) named as3B5,4C8.3F10.5D11,1B12and1G9were obtained. Briefly:first, BALB/c mice were injected with inactivated EHEC0157strain, and then the spleen lymphocytes were fused with myeloma cells SP2/0. After that, indirect enzyme-linked immunosorbent assay (ELISA) and direct agglutination test were applied to screen the MAbs. respectively. LPS of0157and026. extracted and purified by an improved phenol/water method, were served as coating antigens. The result of indirect ELISA showed that all of the six MAbs could react against EHEC0157whole cell but not against EHEC026whole cell at all. moreover,3B5,3F10,4C8and5D11, could respond to LPS of EHEC0157rather than EHEC026. Similarly, the result of direct agglutination test showed that each of the six MAbs reacted well with the whole cells of EHEC0157but not the EHEC026cells. Therefore,3F10,5D11and1G9hybridoma were selected to prepare ascite fluids, being used later in indirect ELISA, direct agglutination test and Western-blot. The titers of the three MAbs of ascite fluids against the whole cells of EHEC0157were equal to3.2X105. whereas the titers of MAb3F10and MAb5D11of ascite fluids against LPS of EHEC0157were6.4X105and1.6X105.determined by indirect ELISA; The titers of3F10and1G9, against the pyrolysis bacterins of EHEC0157were1:50and1:1000, respectively in the direct agglutination test. The results of Western-blot demonstrated the specificity of3F10and5D11to the LPS antigens of EHEC0157. In addition, the above three MAbs were characterized as IgM isotypes, and had no cross-reactions with Salmonella, Pasteurella or other tested non-0157STEC strains, indicating that they had an acceptable specificity.The MAb3F10was futher purified with euglobulin, and its concentration post-purification was8.2mg/mL. Polystyrene beads was coated with3F10, the concentrations of the MAb spre-and post-coupling reaction were determined using BCA protein kit. The concentration of MAb on the immunomagnetic beads was62.6μg/mg for3F10. We determined the O157:H7colony forming units (CFUs) and recovered from the contaminated water pre-and post-absorbing by the immunomagnetic beads, and the absorption rate was54.7%. In conclusion, the immunomagnetic beads based on the MAbs exhibited a satisfied appearance in absorbing the target bacteria and removing the inhibitors from samples for polymerase chain reaction (PCR). The pathogenic bacteriain artificially contaminated samples absorbed by the immunomagnetic beads could be used as templates immediately in multiplex PCR and the sensitivity reached up to105CFU/g while the sensitivity is only106CFU/g if the samples were not absorbed by the immunomagnetic beads. The developed method is simple operating and time-saving, and could be used to screen a large scale of samples rapidly.