Abstract. Seven monoclonal anti-zeatin riboside antibodies were characterized by radioimmunoassay (RIA) and found to measure femtomole (10-15 M) quantities (-20 pg) of this cytokinin. The antibodies had different measuring ranges defined by the linear portion of the logit/log plots; slopes and intercepts of the line varied considerably between the antibodies. Competitive binding trials against cis-zeatin riboside (cZR), dihydrozeatin riboside (diHZR), zeatin (Z), and isopentenyl adenosine (iPA) showed differences among the seven antibodies in their cross-reactivities towards these structurally related cytokinins. It was possible to combine selected antibodies to provide a mixture with a predictable measuring range and cross-reactivity; the ability to prepare a highly specific reagent in this manner with well-defined reactivity was noted and differences between monoclonal antibody and polyclonal antiserum probes for measurement of cytokinins were discussed.Cytokinins play important roles in many plant physiological processes including the regulation of growth and development. Plant hormone research has been hindered by the lack of specific and sensitive methods for qualitative and quantitative assays, in part because cytokinins and other growth regulators
Sixteen monoclonal antibodies against isopentenyl adenosine (iPA) were developed and characterized for reactivity towards this cytokinin and structurally related molecules by use of a competition fluorescence enzyme immunoassay. Antibodies with suitable affinity and specificity were used in an immunoassay to detect and quantify isopentenyl adenosine. Logit/log plots of fluorescence vs pmol of the cytokinin indicated that the assay had a measurement range of 0.03–256 pmol (10–8500 pg) with high linearity (r =–0.98). This competition fluorescence enzyme immunoassay showed that three antibodies cross‐reacted with N‐6‐benzyladenine and its riboside: cross‐reactivity with dihydrozeatin and its riboside, cis‐zeatin, trans‐zeatin riboside, adenine and adenosine was minor. When an immunoaffinity‐HPLC technique was used to measure the cross‐reactivity of two of the antibodies in the presence of known quantities of multiple cytokinins, iPA was bound in preference to the other cytokinins. One of the antibodies was used to quantify this cytokinin in developing wheat (Triticum aestivum L. cv. Stephens) seeds and in wheat seeds spiked with known amounts of certain other cytokinins. The use of these antibodies in immunoassay in combination with HPCL for quantification of iPA in plant extracts is discussed.
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