Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts

Abstract

Two radioimmunoassays have been developed which allow the parallel quantitation of free as well as conjugated natural (+)-abscisic acid (ABA) directly and separately, in unpurified plant extracts. The differential specificity of antisera has been achieved by coupling ABA through C₁ (for total ABA determination) or C₄ (for free ABA determination), respectively, to proteins to obtain the immunogenic conjugates. Compounds structurally related to ABA, such as, dihydrophaseic acid or phaseic acid, do not interfere with either of the assays, even when present in more than ten-fold excess. Other related compounds, such as, violaxanthin or xanthoxin, do not cross react at all. Both antisera respond to (+)-ABA but show very low immunoreactivity with (-)-ABA. As little as 27 pg of ABA (serum for free ABA) or 47 pg (serum for total ABA) may be detected and the measuring ranges are from 0.2–8 and 0.2–30 pmol, respectively. Average recoveries are greater than 99%. Using these assays, more than 100 samples can be assayed for free and conjugated ABA per day. Levels of free ABA, as determined by radioimmunoassay (RIA), correlated well with those reported in the literature. Levels of conjugated ABA were found to be generally higher than previously reported for ABA after alkaline hydrolysis of the extracts. Conjugated ABA accumulates during aging of leaves and levels of conjugated ABA up to 17-fold higher than those of free ABA have been detected in senescent leaves of Hyoscyamus niger L. Evidence was obtained for the presence of ABA conjugates other than the glucose ester in some plants.