A novel immunologic method using antigen-antibody reactions and electrochemical measurement to monitor the oocyte maturation-inducing hormone 17, 20-dihydroxy-4-pregnen-3-one (DHP) in fish was developed. The electrode-based immunologic assay (EBIA) system was based on the competition between DHP and an acetylcholinesterase (AChE)-labelled DHP for a limited number of ant-DHP binding sites. DHP in a sample solution and acetylcholine esterase-labelled DHP were added to the anti-DHP-coated reaction vessel to promote a competitive assay. After one-hour incubation, the vessel was drained and washed five times with a washing buffer. Then, the reaction vessel was incubated with the substrate, acetylthiocholine, for a further 1.5 h. The Pt-Ir electrode was then immersed in the reaction vessel and the current generated at +650 mV at the electrode was used as the analytical signal of the EBIA system. A good correlation was observed between the output current and DHP concentration (1.95-250 pg ml-1) using this method. Optimal system performance was achieved under the following conditions: temperature 26°C, ATCh concentration 10mM. One measurement could be completed within 5 min using the microelectrode and the total assay time, including sample preparation, was less than 3 h. The system was applied to monitor DHP in spawner (rainbow trout) during one month. There was a close correlation between our proposed system and the conventional method. The system can be used for a more rapid determination of the time of ovulation, compared with the conventional method.
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Document Type: Research Article
Faculty of Marine Science, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo 108-8477, Japan
Faculty of Marine Technology, Tokyo University of Marine Science and Technology, Etchujima, Koto-ku, Tokyo 135-8533, Japan
February 1, 2011
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