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

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N-6-benzyladenosine, N-6-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05-70 pmol for N-6-benzyladenine and 0.01-20 pmol for the N-6-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N-6-benzyladenine and N-6-(meta-hydroxybenzyl)adenine and their corresponding N-9-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-H-3]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N-6-benzyladenine and N-6-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solanum 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 N-6-benzyladenine, N-6-(meta-hydroxybenzyl)adenine, and their N-9-glycosides in these tissues.