Immunoaffinity chromatography of abscisic acid combined with electrospray liquid chromatography-mass spectrometry |
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Authors: | Hradecká Veronika Novák Ondrej Havlícek Libor Strnad Miroslav |
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Affiliation: | Laboratory of Growth Regulators, Palacky University & Institute of Experimental Botany ASCR, Slechtitel? 11, CZ-783 71 Olomouc, Czech Republic. hradeckav@post.cz |
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Abstract: | Polyclonal antibodies with high specificity for C1-immobilised (+)-cis,trans-abscisic acid (ABA) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of an immunoaffinity chromatography (IAC) gel. The detection limit of the ELISA was approximately 4.6x10(-10)mol/L. Sensitive electrospray liquid chromatography-mass spectrometry (LC-ESI-MS) methods were also developed with detection limits below 0.1x10(-12)mol. The IAC allowed quick, single-step processing of samples prior to the analyses. The LC-ESI-MS and LC-ELISA techniques were used for comparative estimation of endogenous ABA levels in immunoaffinity purified extracts of normal and water-stressed Nicotiana tabacum L. leaves. The analytical approaches were validated using deuterium- and tritium-labelled internal standards, respectively. The IAC method was found to be highly effective, sensitive and convenient for isolating the target analyte from plant material. |
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