Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 424

Radioactively labeled Ag (Ag*) to bind with Ab is used to form the specific
Ag*-Ab complex in RIA, following which the analytes are quantified by measure-
ment of radioactivity. In the assay, special security measures should be taken in
the assay because radioisotopes are used (Fu et al. 2011 ). RIA is a highly sensi-
tive method, which can detect target molecules at nmol or pmol lever (Tarkowski
et al. 2009 ). After mAbs instead of pAbs were used, one of the major objectives in
developing RIA methods was to reduce the purification procedures prior to quan-
tification, thus some RIA systems without complex purification steps for ABA
analysis were developed (Weiler 1979 ). Because RIA is relatively quick, it is still
used in ABA assay today especially when a large number of small samples to be
processed (Su et al. 2013 ; Thiagarajan et al. 2013 ).
Ag can be labeled with easily assayed enzyme instead of radioisotope in
ELISA. Accordingly, ELISA combines the specificity of Ag-Ab reaction with the
sensitivity of enzymic assays and provides a sensitive method with lower detection
limit for ABA analysis. Weiler ( 1982 ) developed a solid-phase ELISA for abscisic
acid, and abscisic acid conjugates at fmol level (detection limit: 50-60 fmol). Only
little procedural effort was required for the assay which can be completed within
6 h. After that, several ELISA assays including direct ELISA and indirect ELISA
for free and conjugated ABA have been reported (Ross et al. 1987 ; Norman et al.
1988 ; Cahill and Ward 1989 ; Zhang et al. 1991 ). Two solid-phase competitive
ELISA systems were also developed for detecting free and bound ABA (Blintsov
and Gusakovskaya 2006 ). Currently, ELISA kits are commercially available from
biotechnology companies and research institutions. Compared with RIA, ELISA
is less expensive and easier to set up despite the common problem of cross-reac-
tion with structurally similar compounds; moreover, the hazards of RIA associated
with the radioactive waste disposal can be excluded (Tarkowski et al. 2009 ).
Immunosensors are affinity ligand-based solid-state biosensor devices in
which the immunochemical reaction is coupled to a transducer. The fundamental
basis of all immunosensors is also the specificity of the molecular recognition of
Ag and Ab to form a stable complex. In contrast to traditional immunoassays of
RIA and ELISA, modern transducer technology enables the label-free detection
and quantification of the immune complex (Luppa et al. 2001 ). Electrochemical
immunosensor, one of the commonly used immunosensors, combines the sensi-
tivity of electroanalytical methods with the inherent bioselectivity of the biologi-
cal component (e.g., antibody or aptamer). These immunosensors presented good
specificity and high sensitivity and have been successfully applied to ABA analy-
sis (Li et al. 2008 , 2010 ; Wang et al. 2009 ). In addition, immobilization protocols,
biosensor regeneration, signal amplification, equipment miniaturization, and anti-
body (or aptamer) properties are considered in the development and applications
of these immunosensors. For example, to develop an amperometric immunosen-
sor for ABA, an approximate 10-nm-thick gold layer was first sputtered uniformly
onto the electrode surface, and then gold nanoparticles were chemically grown
directly on the gold layer for antibody adsorption by immersing the electrode
into H 2 AuCl 4 solution (Wang et al. 2009 ), or an anti-ABA antibody was adsorbed
directly on a porous nanogold film (Li et al. 2008 ).