caa a22 4500 46317173X CHVBK 20180406164815.0 cr unu---uuuuu 170326e20070301xx s 000 0 eng 10.1007/s00344-006-0045-2 doi (NATIONALLICENCE)springer-10.1007/s00344-006-0045-2 New Techniques for the Estimation of Naturally Occurring Brassinosteroids [Elektronische Daten] [Jana Swaczynová, Ondřej Novák, Eva Hauserová, Květoslava Fuksová, Miroslav Šíša, Ladislav Kohout, Miroslav Strnad] 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. Springer Science+Business Media, Inc., 2007 Antibodies nationallicence Arabidopsis nationallicence Brassinosteroids nationallicence Enzyme immunoassay nationallicence HPLC-MS nationallicence Rape pollen nationallicence Swaczynová Jana Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Novák Ondřej Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Hauserová Eva Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Fuksová Květoslava Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Šíša Miroslav Department of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic aut Kohout Ladislav Department of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic aut Strnad Miroslav Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut Journal of Plant Growth Regulation Springer-Verlag; www.springer-ny.com 26/1(2007-03-01), 1-14 0721-7595 26:1<1 2007 26 344 https://doi.org/10.1007/s00344-006-0045-2 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00344-006-0045-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Swaczynová Jana Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Novák Ondřej Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Hauserová Eva Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Fuksová Květoslava Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 700 1- Šíša Miroslav Department of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic aut NATIONALLICENCE 700 1- Kohout Ladislav Department of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic aut NATIONALLICENCE 700 1- Strnad Miroslav Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, 783 71, Olomouc, Czech Republic aut NATIONALLICENCE 773 0- Journal of Plant Growth Regulation Springer-Verlag; www.springer-ny.com 26/1(2007-03-01), 1-14 0721-7595 26:1<1 2007 26 344 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer