STANDARDIZATION AND EVALUATION OF IMMUNOLOGICAL DETECTION METHOD FOR SHIGA TOXIN EXPRESSING-ESCHERICHIA COLI ISOLATES

AC33

Rocha, LB; Guth, BEC; Piazza, RMF

Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brasil; Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP, São paulo, Brasil

Introduction: STEC infection is an important food borne pathogens that may cause a wide range of disease symptoms, since an asymptomatic event until hemolytic-uremic syndrome and hemorrhagic colitis. The main virulence factors for STEC, the Shiga toxins (Stx) are responsible for protein synthesis inhibition. Numerous assays for it diagnosis have been developed and some of the evaluated kits showed variability in sensitivity and specificity when tested by different reference laboratories. The commercially available immunological kits offered by different companies are economically unaffordable in developing countries. Objectives: The aim of this study was the standardization and evaluation of a specific, sensitive and low cost diagnostic method for Shiga toxins detection. Methods: The definition of a growth media that induce better expression and production of Stx was determined: STEC strains were grown at 37ºC for 24 h in eight different media and toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in the pellet and/or supernatants of cultures in the presence or absence of ciprofloxacin or novobiocin. Besides, selected monoclonal antibodies supernatants were purified by affinity chromatography, isotyped and characterized. Then a capture ELISA using polyclonal and monoclonal antibodies anti-Stx1 and anti-Stx2 were developed. For assay standardization, different concentrations of policlonal and monoclonal antibodies were tested. Also, different dilutions of mouse anti-goat peroxidase conjugated and plates were tested. Results: The results showed that the most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the production is detected in the supernatant. The monoclonal anti-Stx1 and anti-Stx2 antibody was subcloned several times and both were classified as IgG1. Anti-Stx1 antibody was able to recognize the A subunit of both toxins, besides showing higher affinity constant than anti-Stx2 antibody. Thus, the capture ELISA was standardized using 250 mg/ml of the IgG enriched fraction of rabbit anti-Stx1 and anti-Stx2 sera for coating and 2.5 mg/ml of monoclonal anti-Stx1 antibody for the capture of the toxin. After development of the assay with 149 isolated (between positive and negative strains) the capture ELISA showed 80.7% of sensitivity, 100% of specificity and 93% of efficiency. Conclusion: These results indicated the capture ELISA had the sensitivity similar to those in the literature, but isolates expressing-Stx2 were not recognized, probably due anti-Stx1 specificity, as it was able to recognized the A subunit of both toxins only in denaturation conditions, which lead us to the improvement of this method in order to increase the accuracy of the immunoassay.