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Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine
Department of Chemistry, Biotechnology and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-24 Korimoto, Kagoshima, Kagoshima 890-8580, Japan
Genenet CO., LTD. 5-22-8 Tanotsu, Higashi-ku, Fukuoka 813-0034, Japan
Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyou-machi, Nagasaki 852-8521, Japan
Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan
* Author to whom correspondence should be addressed.
Received: 10 December 2012; in revised form: 9 January 2013 / Accepted: 5 February 2013 / Published: 7 February 2013
Abstract: An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv) clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg) but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.
Keywords: morphine; narcotic; hapten; single-chain Fv (scFv); recombinant antibody technology
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Matsukizono, M.; Kamegawa, M.; Tanaka, K.; Kohra, S.; Arizono, K.; Hamazoe, Y.; Sugimura, K. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine. Antibodies 2013, 2, 93-112.
Matsukizono M, Kamegawa M, Tanaka K, Kohra S, Arizono K, Hamazoe Y, Sugimura K. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine. Antibodies. 2013; 2(1):93-112.
Matsukizono, Miho; Kamegawa, Mariko; Tanaka, Koichi; Kohra, Shinya; Arizono, Koji; Hamazoe, Yuta; Sugimura, Kazuhisa. 2013. "Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine." Antibodies 2, no. 1: 93-112.