Monoclonal antibodies against the plant cytokinin isopentenyl adenosine

Abstract

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.

Hybrid Hybridoma Cell Lines Which Express Anti-Zeatin Riboside, Anti-Abscisic Acid, and Hybrid Antibodies

“…With few exceptions (14), quantification by radioimmunoassay or ELISA is preceded by isolation and partial purification of the hormones from biochemically complex plant extracts (1,5,13). The use of immobilized antihormone antibodies in an immunoaffinity matrix has simplified the isolation of certain plant hormones (2,6) and provided greater specificity in the purification procedure.…”

“…Polyclonal and monoclonal antibodies have been developed and used for quantification of a variety of plant growth regulators (3,4,11,13,16). With few exceptions (14), quantification by radioimmunoassay or ELISA is preceded by isolation and partial purification of the hormones from biochemically complex plant extracts (1,5,13).…”

The Development of an Indirect Enzyme Linked Immunoassay for Abscisic Acid

“…A number of methods based on RIA2 or ELISA have been developed (7,8,10,12,14). Many of these assays involve polyclonal antibodies, where the antisera contain multiple populations of antibodies with varying specificity.…”

“…Many of these assays involve polyclonal antibodies, where the antisera contain multiple populations of antibodies with varying specificity. More recently, monoclonal antibodies have been used in assays for plant hormones (7,8,10,12). Use of monoclonal antibodies should eliminate much of the variability due to crossreactivity that is observed with polyclonal antisera.…”

“…Most ELISAs reported to date for the various PGRs have used the direct method of ELISA (4, 13). There are also several reports of an indirect method for cytokinins (10) and ABA (12), utilizing BSA 688 GBq mmol-') was methylated with ethereal diazomethane according to the methods of Schlenk and Gellerman (9).…”