Development of an immunochromatographic test strip for detection of cholera toxin

Abstract

Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.

Figure 1

Reactivity of CT-IC with purified recombinant CT and LT. 0.1 mL of serial diluted purified recombinant CT (a) or LT (b) was applied to the test strips. After 15 minutes, development of red color at position for test (T) or control (C) lines was monitored. Concentrations of samples applied are indicated on right side of each strip. The “+++”, “++”, “+”, or “−” symbols are placed on the left side of the strips developing “strong,” “medium,” “faint,” or “no” bands at test lines, respectively.

Figure 2

Ability of CT-IC to detect the toxigenic V. cholerae strains. Fifteen ct gene-positive V. cholerae isolates and 5 ct gene-negative V. cholerae isolates were cultured under AKI-SW condition, and then obtained supernatants of each culture were examined by quantitative Bead-ELISA specific for CT (a) or CT-IC (b). For quantitative analysis, data are means ± SD of values from three independent experiments. UD: undetectable. For IC, the “+++”, “+”, or “−” symbols were placed on the left side of the strips developing “strong,” “faint,” or “no” bands at test lines, respectively. T: test line, C: control line.

Figure 3

Reactivity of CT-IC with non-V. cholerae strains. Twelve ETEC isolates (a) and 7 V. parahaemolyticus isolates (b) were cultured under AKI-SW condition, and then obtained supernatants of each culture were examined by CT-IC. The “+” or “−” symbols were placed on the left side of the strips developing “faint” or “no” bands at test lines, respectively. T: test line, C: control line.