Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N⁶-benzyladenosine, N⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N⁶-benzyladenine and 0.01–20 pmol for the N⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N⁶-benzyladenine and N⁶-(meta-hydroxybenzyl)adenine and their corresponding N⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N⁶-benzyladenine and N⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N⁶-benzyladenine, N⁶-meta-hydroxybenzyl)adenine, and their N⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N⁶-benzyladenosine