Improvements in the microtitre G_M1 ganglioside enzyme‐linked immunosorbent assay for Escherichia coli heat‐labile enterotoxin

Abstract

A variant of the microtitre GM1-ELISA for Escherichia coli heat-labile enterotoxin was studied. The test was improved by both reducing the assay time from 2 1/2 d to 8 h and by determining the most appropriate GM1 coating concentration. Coating the plates with greater than or equal to 3 micrograms of GM1/ml yielded a maximal sensitivity and ensured a linear relationship between the enterotoxin concentration and the extinction observed when using the final assay-procedure. Thus an optimal accuracy was obtained. This ELISA was 4- to 8-times more sensitive than the Vero cell monolayer assay. The sensitivity of this ELISA and of the chinese hamster ovary cell monolayer assay were identical.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

“…Microtiter GM1 ganglioside methods are widely employed for the detection of LT toxin (Gustafsson & Molby 1982, Ristaino et al 1983, Svennerholm & Wiklund 1983, Sen et al 1984, Bongaerts et al 1985. Thus, the sensitivity and specificity of the capture immunoassay standardized by Menezes et al (2003) was compared with GM1 ELISA.…”

“…Then, several immunological assays have been described, mainly the protocol that LT is captured by ganglioside GM1, its receptor in the host cell (Gustafsson & Molby 1982, Ristaino et al 1983, Svennerholm & Wiklund 1983, Sen et al 1984, Bongaerts et al 1985. Additionally, agglutination assays were developed for LT detection (Speirs et al 1991).…”

Enzyme-linked immunosorbent assay for Aeromonas hydrophila hemolysins

“…The medium reported by Evans et al [11] was used for production of cholera-like enterotoxin. The culture was clarified by centrifugation at 10 000 x g for 30 min at 4 ° C. Cholera-like enterotoxin was a examined by ganglioside GM1-ELISA [16]. Ganglioside GM1, purified from human brain, was a gift from M. Iwamori, Faculty of Medicine, University of Tokyo.…”

Evaluation of experimental conditions for quantification of LT produced by human derived enterotoxigenic Escherichia coli strains

“…Similarly, cELISA relies on the specific recognition of surface-exposed epitopes by polyclonal or monoclonal anti-LT or anti-CT antibodies (8,15,30). Both methods have been successfully used to detect LT production by different ETEC strains but only GM1-ELISA was previously evaluated in regard to factors that influence its sensitivity (5).…”

“…Several LT-extraction procedures have been reported including the use of polymyxin B, Triton X-100, lincomycin, mitomycin C and sonic disruption, but the definition of the best approach based on a comparative quantitative analysis has not been performed (5,8,20,23). In this study we evaluate two aspects concerning quantification of LT produced by ETEC strains: the experimental parameters affecting the optimal performance of a LT quantitative method (cELISA) and a comparison of different techniques employed for releasing LT produced by ETEC strains.…”