Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 423

anti-ABA antibodies because many phytohormonal analogs, metabolites, and other
structurally similar compounds present in plant sample. The antibody preparation
step is the key factor for establishment of immunoaffinity purification methods
for ABA. To improve the durability and reusability of immunoaffinity purification
methods, a pre-cleanup process with C 18 -based SPE (Hradecká et al. 2007 ; P

ík
et al. 2009 ; Novák et al. 2008 ; Simersk� et al. 2009 ), mixed-mode SPE (Liang
et al. 2012 ), or semi-preparative HPLC (Du et al. 2010 ) was often necessary in the
purification of extracts from real plant tissues. Because of their higher selectivity
but lower throughput than conventional SPE, immunoaffinity purification methods
still have good potential for purification of trace ABA in plant samples of small
quantities.

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21.2.3 Techniques for Analysis

In plant tissues, phytohormones including ABA are present at very low levels
against a background of a wide range of more abundant primary and secondary
metabolites. Despite the application of complex purification steps to separate
the analyte from crude plant extracts, there are still a large number of interfering
metabolites in the samples for final assay, highly selective and sensitive analytical
methods for ABA analysis are still essential. For the determination and quantifica-
tion of ABA, many analytical methods, such as bioassay, immunoassay, biosen-
sor, chromatography, and chromatography/mass spectrometry, have been adopted.
Many advanced analytical techniques, such as single-cell capillary electrophore-
sis, nanoelectrospray, and quantum dots, are increasingly being explored in the
development of ABA analytical methods. In this section, two types of commonly
used methods including immuno-based methods and chromatographic methods are
summarized.

21.2.3.1 Immuno-based Methods

Immuno-based methods including radioimmunoassay (RIA), enzyme-linked
immunosorbent assay (ELISA) and immunosensor are specific detection meth-
ods based on the specific antibody-antigen (Ab-Ag) binding property. The spec-
ificity of the Ab-Ag interaction is the key factor affecting quantification results.
However, due to the low-affinity and strong cross-reactions of polyclonal antibod-
ies (pAbs) in the presence of high levels of ABA precursors, ABA catabolites or
conjugates, the application of immunoassay techniques for the quantitative analy-
sis of ABA were limited initially. In 1980s, the introduction of monoclonal anti-
bodies (mAbs) has provided attractive alternatives to the traditional immunoassays
of ABA by reducing the need for purification and speeding up analysis for a large
number of samples (Mertens et al. 1983 ; Weiler 1984 ; Harris and Dugger 1986 ,
Harris et al. 1988 , Leroux et al. 1985 ).