Evaluation of a ganglioside immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin (original) (raw)
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Journal of Clinical Microbiology, 1982
Human and porcine enterotoxigenic strains of Escherichia coli were cultivated in tryptone-yeast extract medium or brain heart infusion broth and tested for production of heat-labile enterotoxin by the GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) and the Y1 adrenal cell assay. When testing for enterotoxigenicity by the GM1-ELISA technique, homologous antisera for human and porcine heat-labile enterotoxins had to be used to detect enterotoxigenic strains of human and porcine origin, respectively. This observation indicates a serological difference between the heat-labile enterotoxins produced by human and porcine strains. Furthermore, brain heart infusion broth was found to have an inhibitory effect on detection of enterotoxin both in the GM1-ELISA and in a toxin-binding modification of the Y1 adrenal cell test, but not in the conventional adrenal cell assay.
Journal of Clinical Microbiology, 1983
A new method has been developed for demonstration of heat-labile (LT) enterotoxin produced by Escherichia coli. This method is based upon the release of LT from bacteria grown directly onto agar plates which have been coated with ganglioside GM1. Toxin bound to the GM1 solid state is subsequently demonstrated by means of a three-step immunoenzymatic procedure in which enzymesubstrate reactions are visualized as dark spots in agarose. When analyzing LT production from 105 E. coli strains, results obtained by this procedure (GM1-ELISPOT) correlated well with those of the GM1 enzyme-linked immunosorbent assay (GM1-ELISA); in no instance were any false-positive reactions observed when highly specific monoclonal antibodies against LT were used. Easy to perform, the GM1-ELISPOT allows demonstration of LT within 24 h after inoculation of the plates, and large numbers of specimens can be screened at the same time without the need of any special equipment. Thus, this new method should meet the requirements of any diagnostic laboratory.
Journal of Clinical Microbiology, 1988
Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared. In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes. As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes. The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes. In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes. The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml. Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, ...
GM1 erythroimmunoassay for detection and titration of Escherichia coli heat-labile enterotoxin
Journal of Clinical Microbiology, 1986
A GM1 ganglioside erythroimmunoassay for the detection of heat-labile Escherichia coli enterotoxin (LT) was developed for use in poorly equipped laboratories in developing countries. This assay is based on the immunological similarity between Vibrio cholerae toxin and LT and uses cholera toxin antiserum and sheep anti-rabbit immunoglobulin covalently coupled to sheep erythrocytes as conjugate. This assay has the following advantages over other currently available techniques: the reagents it uses are stable, in particular, tanned and sensitized sheep erythrocytes; GM1 ganglioside is commercially available; erythro-adsorption can be read with the naked eye; the test can be completed in 1 day; and as little as 4 ng of V. cholerae toxin or LT per ml can be detected accurately. The GM1 ganglioside erythroimmunoassay showed good quantitative and qualitative correlation with the Vero cell assay and the conventional GM1 enzyme-linked immunosorbent assay. The GM1 ganglioside erythroimmunoass...
Antibody chimera technique applied to the detection of Escherichia coli heat-labile enterotoxin
Journal of Immunological Methods, 1987
Covalently prepared chimera antibodies were tested in a ganglioside GM1 erythro-immunoassay (CERIA) for E. coli heat-labile enterotoxin (LT) detection. The antibody specific for LT was conjugated with a polyclonal antibody specific for sheep erythrocytes. The assay is based on the specific binding of LT to polystyrene-adsorbed GM1 and subsequent erythro-adsorption via chimera antibody by which the bound toxin is visualized. Enterotoxin titers determined with this CERIA method were similar to those obtained with the Vero cell assay and with ELISA. 5 ng of cholera toxin/ml may be detected with the assay. The CERIA, as described, may be used either qualitatively or quantitatively and is well suited for routine laboratory diagnosis of LT in a culture supernatant of E. coll.
Journal of Clinical Microbiology, 1980
We have developed a microtiter enzyme-linked immunosorbent assay method for detecting the heat-labile enterotoxins of Vibrio cholerae and Escherichia coli using GM1 ganglioside as the base coat. This method compares favorably with a similar assay using anticholera toxin as the base coat, and with the Y1 adrenal cell assay. The assay should be useful in detecting enterotoxin production in E. coli and vibrios (including non-agglutinating Vibrio), in quantitating the toxin, and in determining binding properties of enterotoxins to ganglioside. The assay can also be used to quantitate antibodies which block the attachment of the toxin to the ganglioside.
Detection of Escherichia coli enterotoxins in stools
Infection and immunity, 1980
We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method.
Infection and immunity, 1977
Acute- and convalescent-phase sera from 132 students attending a university in rural Mexico were assayed for antibody against the heat-labile enterotoxin (LT) of Escherichia coli by neutralization of LT activity in the Y-1 adrenal cell assay and by passive immune hemolysis of LT-sensitized sheep erythrocytes. The two titration methods produced comparable results with respect to antitoxin responses detected. An inverse relationship was found between acute geometric mean antitoxin titer and the occurrence of diarrhea associated with LT-producing E. coli, especially in newly arrived students from the U.S.A. A significant correlation (P less than 0.00 5) was found between a rise in antitoxin titer detectable by the passive immune hemolysis technique and diarrhea with LT-producing E. coli isolated. Thus, humoral antitoxin titers appear to be a useful indicator of immune status with respect to enterotoxigenic (LT) E. coli diarrhea.
Assay for heat-labile Escherichia coli enterotoxin using sandwich erythroimmunoassay
Medical Microbiology and Immunology, 1987
We investigated the possibility of using a sheep erythrocyte-antibody conjugate as reagent in a sandwich erythroimmunoassay (SERIA) procedure to detect and titrate Escbericbia coli heat-labile enterotoxin (LT) with the naked eye. In this assay, which is based on the immunological similarity between Vibrio cbolerae toxin (CT) and LT, rabbit anti-CT IgG was used as immunosorbent, and sheep erythrocytes, sensitized with the rabbit anti-CT antibodies, were used as indicator. The sensitivity of the test was demonstrated by a comparative study using an enzyme-linked immunosorbent assay (ELISA). The resuits obtained by SERIA with 130 samples correlated well with those of a Vero cell assay and a GM 1-EL1SA. The test is easy, relatively cheap and as sensitive as other standard techniques; it is particularly suited for field laboratories, especially in tropical countries, and a large number of strains may be examined daily.
Brazilian Journal of Microbiology, 2007
The heat-labile toxin (LT) is a key virulence-associated factor associated with the non-invasive secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains either in humans or domestic animals. Several LT detection methods have been reported but quantification of the toxin produced by wild-type ETEC strains is usually performed by the GM1 ganglyoside enzyme-linked immunosorbent assay (GM1 ELISA). In this study we conducted the optimization of an alternative LT-quantification method, the antibody-capture ELISA (cELISA). Detailed analysis of the appropriate dilutions of capture and detecting LT-specific antibodies significantly improved the sensitivity of the method. Additionally, testing of different LT extraction techniques indicated that sonic disruption of the bacterial cells enhanced LT recovery yields, in contrast to the usual procedure based on addition of polymyxin B to the culture medium as well as extraction methods based on chloroform or Triton X-100. Moreover, the present data indicate that performance of the LT extraction method based on polymyxin B treatment can vary among wild ETEC strains.