Electrode-based immunologic assay system to monitor oocyte maturation-inducing hormone in fish

Abstract

A novel immunologic method using antigen-antibody reactions and electrochemical measurement to monitor the oocyte maturation-inducing hormone 17, 20β-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.

Keywords:

• biosensor
• immunoassay
• electrode
• fish
• hormone
• ovulation

Acknowledgements

This research was supported in part by a Grant-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science, and Technology. We wish to thank Professor Kiyoshi Asahina of Nihon University for helpful discussion.