Good correlation between enzyme-linked immunosorbent assay and gas chromatographic analysis of abscisic acid in apple organs.
Good correlation between enzyme-linked immunosorbent assay and gas chromatographic analysis of abscisic acid in apple organs.
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苹果器官中脱落酸的酶联免疫吸附测定与气相色谱分析之间具有良好的相关性。
DOI：
10.2503/jjshs.58.819
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
S. Yamaki
中科院分区：
文献类型：
--
作者：
J. Soejima;Masako Watanabe;T. Moriguchi;S. Yamaki
检测苹果各器官中所含的内源脱落酸（阿坝）以确定测定的量之间是否存在良好的相关性，通过使用电子捕获检测器（GLC-ECD）的气相色谱法和使用单克隆抗体的酶联免疫吸附测定法对应于sABA的保留时间的清晰峰出现在GLC-100上。在用以下步骤制备样品后，甲酯化样品的ECD分析：游离和缀合阿坝提取物的MeOH提取、乙酸乙酯分配和薄层色谱（TLC）分离。从分析的各个器官的峰包括内源游离和共轭阿坝在苹果器官，并证实主要是阿坝的气相色谱-质谱（GC-MS）鉴定。经上述纯化步骤制备样品后，用ELISA-MA法测定的苹果茎、叶和果实内源阿坝含量与GLC-ECD法测定的结果一致。对于ELISA-MA分析，可以省略对叶或茎样品的一些纯化步骤。此外，由于ELISA-MA与阿坝的结合亲和力非常高，因此可以分别在茎、叶和果实的仅50、50和200μg鲜重的样品中检测到ABA。因此，ELISA-MA方法是一个很好的工具，以确定内源性阿坝含量的微观尺寸的组织样品，如果适当的预纯化步骤，建立了个别样品类型。
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.