Detection of Escherichia coli producing Lt-I enterotoxin by capture immunoassay

MENEZES, C.A.(1), LIMA, F.A.(1), FERNANDES, I.(2), AMIANTI, J.(1), KOGA, P.C.M.(1), TRABULSI, L.R.(1), PIAZZA, R.M.F.(1)

(1)Laboratório Especial de Microbiologia, (2)Laboratório de Imunopatologia, Instituto Butantan, São Paulo, SP, Brazil.

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen responsible for traveler’s diarrhea and causes more than 700,000 childhood deaths per year due to diarrhea in third-world countries. ETEC strains cause diarrhea through the action of the enterotoxins ST and LT. These strains can express ST-I , LT-I , or both. Detection of ETEC has been long relied on detection of the enterotoxins LT and/or ST through biological methods, immunoassays and molecular diagnostic techniques. The aim of present data was standardized an immunoassay for detection of diarrheagenic E. coli producing LT-I toxin. Cell fusion with B cells from lymph nodes of immunized mice with LT-I toxins resulted in one positive and stable clone, characterized as IgG2b with binding capacity of 8,4 x 10⁴ molecules to LT-I and dissociation constant of 3 x 10^-6M. This monoclonal antibody was able to inhibit 50% the ability of LT-I to bind to GM1 receptor. We also raised rabbit´s antisera to LT-I toxin. The IgG enriched fraction of this antisera showed specificity and was able to inhibit up to 85% of the binding of LT-I to its receptor. The capture assay developed using the IgG enriched fraction of anti-LT-I antisera and IgG2b anti-LT-I monoclonal, allowed a clear distinction between E. coli LT-I - producing and non-producing. Statistical analysis showed that in capture immunoassay the variances between two groups are significantly different, and these results suggests that sensibility of capture immunoassay is higher than ELISA-GM1 and Vero cells assay.

CORRESPONDENCE TO:

PIAZZA, R.M. F., Laboratório Especial de Microbiologia, Instituto Butantan, Avenida Vital Brasil 1500, 05503-900, São Paulo, SP, Brasil, Email: roxane@usp.br