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Development of a cell-based assay for ecdysteroid quantification using an early ecdysteroid-inducible gene promoter

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Abstract

Ecdysteroids, primarily 20-hydroxyecdysone (20E) and ecdysone (E), are steroid hormones that regulate various developmental and physiological processes in insects. Commonly, immunoassays are used to quantify ecdysteroid titers of insects. However, the antibodies used in these assays react not only with 20E and E but often also with their inactive reserves and metabolites, and thus require purification before they can be quantified precisely. Here, we developed a simple cell-based method to quantify only the hormonally active ecdysteroids using newly established cells harboring the firefly luciferase gene under the control of the ecdysteroid-inducible promoter of the E75A gene of the silkworm Bombyx mori L. These cells also constitutively expressed the Renilla luciferase gene using the baculovirus ie2 promoter for internal reference. This cell-based method detected hormonally active ecdysteroids with significantly higher sensitivity than their inactive metabolites. Hemolymph ecdysteroid titers, determined using a dual luciferase assay after exposing these cells to crude extracts of B. mori larval and pupal hemolymph, agreed well with the sum of the 20E and E titers, which were quantified individually using a radioimmunoassay after they had been separated by HPLC. Thus, this method is very useful for quantifying the ecdysteroid titers of insects, particularly when the samples contain large amounts of ecdysteroid reserves and metabolites.

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Acknowledgments

This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and Grants-in-Aid for Scientific Research 26292179 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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Author notes

  1. Hitoshi Matsumoto

    Present address: Department of Applied Biological Sciences, Saga University, Honjo, Saga, Saga, 840-8502, Japan

Authors and Affiliations

  1. Division of Insect Science, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki, 305-8634, Japan

    Manabu Kamimura, Hitoshi Matsumoto, Makoto Kiuchi, Yuka Ito & Tetsuro Shinoda

  2. Department of Integrated Biosciences Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8562, Japan

    Haruhiko Fujiwara

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  1. Manabu Kamimura
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  2. Hitoshi Matsumoto
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  3. Makoto Kiuchi
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  5. Haruhiko Fujiwara
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  6. Tetsuro Shinoda
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Correspondence to Manabu Kamimura.

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Kamimura, M., Matsumoto, H., Kiuchi, M. et al. Development of a cell-based assay for ecdysteroid quantification using an early ecdysteroid-inducible gene promoter. Appl Entomol Zool 49, 443–452 (2014). https://doi.org/10.1007/s13355-014-0271-1

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  • DOI: https://doi.org/10.1007/s13355-014-0271-1

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