Open-sandwich immunoassay for sensitive and broad-range detection of a shellfish toxin gonyautoxin

• The V genes of an antibody for a shellfish toxin gonyautoxin were cloned using phage display.
• An open sandwich ELISA was designed with the cloned antibody fragment.
• The assay had more than three orders of magnitude working range while keeping subnanomolar sensitivity.

At present, the analytical method for paralytic shellfish poisoning (PSP) toxins in shellfish is the mouse bioassay (MBA), which is an official method of the Association of Analytical Communities (AOAC [8]). However, the low sensitivity and concerns over the number of live animals required for testing have been cited as the major reason for seeking its replacement. In this report, we employed an open-sandwich immunoassay (OS-IA) to detect gonyautoxin (GTX2/3), a kind of PSP toxins. OS-IA, which utilizes the antigen-induced enhancement of antibody VH/VL interaction, can measure a small molecule antigen in a noncompetitive format. Hence it has a wider working range and shorter measurement time. We isolated anti-GTX2/3 antibody gene from a hybridoma GT-13A by screening a Fab-displaying phage library. Then the vectors for OS-IA were constructed, and examined for antigen concentration-dependency of the VH/VL interaction by OS-ELISA. As a result, in each case, signal intensity increases notably in a wide concentration range (0.1 to >1000 ng mL−1) of free GTX2/3, which was enough to cover its regulation value (80 μg 100 g−1) in many countries. So OS-IA will be widely applicable to detect PSP toxins in shellfish meats and in drinking water.

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