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Journal of Lipid Research, Vol. 47, 1572-1582, July 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
Methods |
* Department of Medicinal Information, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
International University of Health and Welfare, 13-1 Higashikaigan-cyo, Atami-shi, Shizuoka 413-0012, Japan, and Department of Internal Medicine, Keio University, School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo 160-8582, Japan
Published, JLR Papers in Press, April 7, 2006.
1 To whom correspondence should be addressed. e-mail: yanakano{at}pharm.showa-u.ac.jp
Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 ± 5.5 µg/ml (mean ± SD) in healthy women and 6.2 ± 3.6 µg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).
Supplementary key words high-molecular-weight multimer • monoclonal antibody • gelatin-binding protein of 28 kDa
Abbreviations: CRP, C-reactive protein; GBP28, gelatin binding protein of 28 kDa; HMW, high-molecular-weight; POD-IH7, horseradish peroxidase-conjugated IH7 Fab'
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