Abstract
Endogenous abscisic acid (ABA) contained in various organs of apple was examined to determine if there was a good correlation between the amounts determined, both by gas chromatography using an electron capture detector (GLC-ECD) and by enzymelinked immunosorbent assay using monoclonal antibody (ELISA-MA).Clear peaks corresponding to the retention time of sABA appeared on GLC-ECD analysis of methyl esterified samples after preparing the sample with the following steps: MeOH extraction, ethyl acetate partitioning and thin layer chromatography (TLC) separation of free and conjugated ABA extracts. The peaks resulting from the analysis of various organs included endogenous free and conjugated ABA in apple organs and were confirmed to be mainly ABA by gas chromatography-mass spectrometric (GC-MS) identification. This supports the conclusion that ABA can be determined quantitatively by GLC-ECD analysis.Endogenous ABA content of stems, leaves and fruits of apple determined by ELISA-MA coincided with those measured by the GLC-ECD method after preparing samples using the above purification steps. Some purification steps on leaf or stem samples could be omitted for ELISA-MA analysis. Moreover, as the binding affinity of ELISA-MA to ABA is very high, it was possible to detect it in samples of only 50, 50 and 200μg fresh weight of stems, leaves and fruit, respectively. Thus, the ELISA-MA method is an excellent tool to determine the endogenous ABA content in micro-size samples of tissue, if the appropriate pre-purification steps are established for individual sample types.
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