Resumo

EPEC and EHEC are two important diarrheagenic Escherichia coli pathotypes; they are responsible for high rates of morbidity and mortality worldwide.  E. coli secreted protein B (EspB) is amongst the virulence factors involved in their pathogenesis, therefore, a potential target for detection.  The diagnosis is an essential tool either for the treatment of disease or to prevent further outbreaks.  In Brazil, there are no commercial kits for detection of EPEC and EHEC, and then their developments are extreme important.  In this sense, the present study aimed the production and use of anti-EspB monoclonal antibodies for the diagnosis of EPEC and EHEC.  For this, Balb/c mice were immunized with 20 µg of purified EspB.  The mouse with the highest antibody titer was boosted with the same amount of purified EspB without adjuvant four days prior to cell fusion, and then sacrificed by cervical dislocation.  Popliteal lymphnodes were removed aseptically and B lymphocytes were fused with myeloma cells in polyethyleneglycol presence.  Ten days after the fusion, hybridomas were screened for antibody production by ELISA.  The specificity of the hybridomas was evaluated by immuno-dot using supernatant of positive and negative bacterial isolates.  Two hybridomas were selected and subjected to limited dilution and the selected clone was cultivated on a large scale in order to collect the supernatant.  The IgG isotype was characterized by ELISA and purified by affinity chromatography column containing Protein A.  The purified monoclonal antibody was titrated by ELISA using 1.5 µg / mL of EspB and the dissociation constant was calculated.  After the characterization of the monoclonal antibody, detection of EspB was standardized by ELISA using supernatants of bacterial isolates cultivated in Dulbeco's MEM.  We obtained more than 300 secreting anti-EspB hybridomas and by immuno-dot we observed a high specificity of 4D9 clone.  This clone was characterized as IgG2a and its dissociation constant was 2 x 10^{-9 M and} only 1 µg / ml was needed for detection of EspB in the supernatant of the isolates.  These results indicate that this monoclonal anti-EspB is a promising tool for the detection of EPEC and EHEC. Suported by FAPESP.