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New Techniques for the Estimation of Naturally Occurring Brassinosteroids

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Abstract

We have developed enzyme-linked immunosorbent assays (ELISAs) for measuring 24-epicastasterone and related brassinolide analogs, with detection ranges of 0.005 to 50 pmoles. Polyclonal antibodies used in these assays were raised against 24-epicastasterone carboxymethyloxime-bovine serum albumin conjugates and were found to have high specificity for 24-epibrassinosteroids. Natural brassinosteroids (BRs), such as brassinolide and 24-epibrassinolide, exhibited relatively high cross-reactivities with the generated antibodies, whereas other BR analogs with β-oriented hydroxyl groups at C-2, C-3, C-22, and C23 lacked immunoreactivity. Through the use of internal standardization, dilution assays, recovery of authentic [3H]24-epicastasterone, and immunohistograms, the ELISAs have been shown to be applicable for estimating 24-epibrassinosteroid levels in crude plant extracts. To analyze brassinosteroids in tissues from young bean (Phaseolus vulgaris L., cv. Pinto), Daucus carota ssp.sativus plants and Arabidopsis thaliana L. Heynh. seedlings, and rape (Brassica napus L.) pollen, the extracts were fractionated by high performance liquid chromatography (HPLC) and the resulting fractions were analyzed by the ELISA method. Immunohistogram ELISA analysis of HPLC fractions indicated that major peaks of immunoreactivity co-chromatographed with the labeled and unlabeled 24-epibrassinolide. A highly sensitive electrospray ionization mass spectrometry (MS) technique (LOD: 50 fmol) was also developed and the results obtained by the HPLC-ELISA and HPLC-MS approaches were compared.

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Acknowledgments

The authors thank Jarmila Balonová for excellent technical assistance. We would also like to thank Sees-editing Ltd. (http://www.sees-editing Ltd.) for the excellent editing of this manuscript. This work was supported by a grant from the Czech Ministry of Education (No. MSM 6198959216, LCO 6034, Z406605061).

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Authors and Affiliations

  1. Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic

    Jana Swaczynová, Ondřej Novák, Eva Hauserová, Květoslava Fuksová & Miroslav Strnad

  2. Department of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic

    Miroslav Šíša & Ladislav Kohout

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  1. Jana Swaczynová
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  2. Ondřej Novák
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  3. Eva Hauserová
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  4. Květoslava Fuksová
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  5. Miroslav Šíša
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  7. Miroslav Strnad
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Correspondence to Miroslav Strnad.

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Swaczynová, J., Novák, O., Hauserová, E. et al. New Techniques for the Estimation of Naturally Occurring Brassinosteroids. J Plant Growth Regul 26, 1–14 (2007). https://doi.org/10.1007/s00344-006-0045-2

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