Chemiluminescent Enzyme Immunoassay for Measuring Leptin

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  • SEKIGUCHI Satoshi
    Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University
  • KOHNO Hideki
    Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University
  • YASUKAWA Kiyoshi
    Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University
  • INOUYE Kuniyo
    Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University

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Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1–1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.

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