Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples

Abstract

A comparison of DOT-ELISA and Standard-ELISA was made for detection of Vibrio cholerae toxin in culture supernatants of bacteria isolated from human and environmental samples. A total of 293 supernatants were tested in a double blind assay. A correlation of 100 % was obtained between both techniques. The cholera toxin was found in 20 Inaba and 3 Ogawa strains. Positive samples were from seafood (17 samples), potable water (1 sample) and sewage (5 samples). The DOT-ELISA was useful as the standard-ELISA to confirm the presence of cholera toxin in the environmental samples.

Keywords: Cholera; DOT-ELISA; Mexico.

Fig. 1

Analytic sensitivity of a standard ELISA to determine the cholera toxin in supernatants of Vibrio cholerae bacteria. Microtiter plates were coated with GM1 ganglioside and after washing and blocked, a number of wells were supplied with supernatant of no-toxin producing bacteria and then, cholera toxin (0 to 0.1 mg/mL) was added to create a dose–response curve; each concentration was tested by triplicate. The reaction was developed with a rabbit IgG anti-cholera toxin and Goat anti-rabbit IgG alkaline phosphatase conjugate. The horizontal dotted line is the cut off obtained with 21 supernatants of no-toxin producing bacteria. Data of linear regression is show at right

Fig. 2

Analytic sensitivity of a DOT-ELISA to determine the cholera toxin in supernatants of Vibrio cholerae bacteria. Circles nitrocellulose membrane were coated with IgG anti-cholera toxin. After blocked with skimmed milk, a number de circles were incubated with the cholera toxin (0 to 0.1 mg/mL) and supernatants of no-toxin producing bacteria to create a dose–response curve. The reaction was developed with an anti-cholera toxin biotin conjugate and avidin-horseradish peroxidase The colour reaction was developed with 3,3′diaminobenzidine and a brown dot in the circle was considered as a positive reaction while, the colourless were considered as negative. Results of three different supernatants are show in a,b and c lines, while a supernatant without cholera toxin is show in d line