We developed a novel flow injection assay for cortisol based on competitive immunologic reactions, magnetic separation, and electrochemical measurement. The proposed flow assay system was composed of two reaction units. An anti-cortisol antibody was immobilised on magnetic beads and injected into the reaction coil of a competitive reaction unit with a blood sample and a specific quantity of acetylcholinesterase-labelled cortisol (cort-AChE). After reacting in the reaction coil, the sample was separated magnetically using a neodymium magnet. The cort-AChE was detached from the magnetic beads and transferred into the enzyme reaction unit with acetylthiocholine (ATCh). ATCh was hydrolysed by the cort-AChE to produce thiocholine. The thiocholine was quantified downstream by electrochemical detection using a Pt-Ir electrode. The performance of the proposed flow assay system was optimised under the following conditions: pH 7.5, temperature 25°C, flow rate 170 µl min-1, ATCh concentration in the substrate buffer 5 mmol L-1. The output current was well correlated with the concentration of the cortisol standard solution (range: 7.8-500 pg mL-1). The results obtained using the proposed flow method were compared with those obtained using conventional ELISA (correlation coefficient 0.9585 [y = -0.9797 + 1.173(x), n = 11]). These findings suggest that the EFIIA system can be used to analyse cortisol in fish plasma samples.
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Document Type: Research Article
Faculty of Marine Science, Tokyo University of Marine Science and Technology 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
Faculty of Marine Technology, Tokyo University of Marine Science and Technology 2-1-6 Etchujima, Koutou-ku, Tokyo, 135-8533, Japan
February 1, 2011
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