The Determination of Phaseic Acid by Monoclonal Antibody‐Based Enzyme immunoassay

Abstract

A monoclonal antibody PA3‐2‐B3, IgG1 (Λ) is described which specifically recognizes phaseic acid and shows very little cross‐reactivity (0.14%) with abscisic acid or dihydrophaseic acid (0.88%). Based on this antibody, an enzyme immunoassay was developed which displays a linearity range from 15 pg to 3 ng of phaseic acid. Results obtained with this assay agree with those obtained by gas chromatography‐electron capture detection. Using the novel enzyme immunoassay, as well as an established immunoassay for abscisic acid, levels of these two compounds in leaves of Phaseolus vulgaris were determined as a function of plant age, water stress, recovery from stress, and feeding of abscisic acid through the transpiration stream. The production of phaseic acid in a microsomal system from bean leaves was demonstrated. The results show a regulation of the plant's capacity to metabolize abscisic acid to phaseic acid as a function of water stress.

(+)-Abscisic Acid 8′-Hydroxylase Is a Cytochrome P450 Monooxygenase

“…In all of these, the reported activities were very low and the assays difficult to reproduce and thus not amenable to detailed characterization of the enzyme. However, collectively, the data suggest that ABA 8Ј-hydroxylase may be a Cyt P450 monooxygenase (Gillard and Walton, 1976;Gergs et al, 1993;Babiano, 1995).…”

“…There have been several previous reports of in vitro ABA 8Ј-hydroxylating activity using microsomal preparations from eastern wild cucumber endosperm (Gillard and Walton, 1976), bean leaves (Gergs et al, 1993), and chickpea seedlings (Babiano, 1995). In all of these, the reported activities were very low and the assays difficult to reproduce and thus not amenable to detailed characterization of the enzyme.…”

“…ABA degradation can be very rapid; the half-life of radioactive ABA applied through the xylem of detached leaves of maize (Zea mays) and Commelina communis was 42 and 64 min, respectively (Jia et al, 1996), and the turnover of ABA taken up from the medium in maize root tips was greater than 60% in a 1-h period (Ribaut et al, 1996). ABA 8Ј-hydroxylase activity is inducible by its substrate, (ϩ)-ABA, in some tissues (Uknes and Ho, 1984;Gergs et al, 1993;Cutler et al, 1997), and there is evidence that it is also regulated by osmotic stress and phytochrome-dependent signaling pathways (Kraepiel et al, 1994;Cutler et al, 1997).…”

Phaseic Acid, an Endogenous and Reversible Inhibitor of Glutamate Receptors in Mouse Brain

“…The presence of (-)-PA in CSF, choroid plexus and cerebral vascular endothelial cells-To demonstrate where (-)-PA is exactly localized in the brain, a mouse monoclonal antibody made specific to PA was used (12). This antibody cross-reacts with both forms of PA, but does not react with ABA, nor with DPA (12).…”

“…Mouse monoclonal antibody to PA was a kind gift from Prof E.W. Weiler (Department of Plant Physiology, Ruhr-University Bochum, Germany) as previously described (12,34). The antibody to PA was diluted with distilled water to 0.2 mg/ml and used at a 1:50 dilution.…”