The development of monoclonal antibodies against the cytokinin zeatin riboside

Abstract

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

tRNA Is the Source of Low-Level trans- Zeatin Production in Methylobacterium spp

“…Eluates were dried in vacuo, resuspended in 400 l of dimethyl sulfoxide, and diluted with 40 mM ammonium acetate (pH 7.0) to obtain 40-ml preparations. Samples were passed through DEAE-cellulose (DE-52; bed volume, 10 ml; Whatman) connected in tandem to immunoaffinity columns, each containing 0.5 to 1.0 ml of microcrystalline cellulose (Whatman) to which purified clone 16 (anti-trans-zeatin and anti-trans-ribosylzeatin) monoclonal antibody (44) had been conjugated (29). The columns were each equilibrated with 120 ml of immunoaffinity buffer and, after sample application, were washed with an additional 20 ml of this buffer passed through both columns and 10 ml passed through only the immunoaffinity column.…”

“…Cytokinins were eluted from the immunoaffinity columns with 15 ml of methanol and dried in vacuo . Isopentenyladenine (iP) and isopentenyladenosine (iPA) not isolated by low-affinity binding to clone 16 monoclonal antibody were isolated similarly by using the clone 12 broad-range cytokinin monoclonal antibody described by Trione et al (44). Similarly, ciszeatin was isolated by using a monoclonal antibody for this isomer obtained from Gary Banowetz (USDA Agricultural Research Service, Oregon State University, Corvallis).…”

Monoclonal Antibodies to Plant Growth Regulators

“…3). Since mAB were raised against synthetic (+) DHZR-BSA con- Table II (24) for mAB screening, often show less sensitivity and less precision than the immobilized antibody assay technique (J Eberle, unpublished observation).…”

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

“…Culture fluids from cell lines 101 B (IgG 1, IgG2a) and 11 1 C (IgM, IgG2a) were studied further to determine whether the expressed antibodies represented hybrid molecules, separate populations ofantibodies derived from one or the other fusion partner, or a combination.…”

“…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).…”