A quantitative fluorescence enzyme immunoassay for plant cytokinins

An enzyme-linked immunosorbent assay (ELISA) which used 4-methylumbelliferyl phosphate as an enzyme substrate was used to quantify two plant cytokinins. This assay detected as little as 0.03 pmol (approximately 10 pg) of cytokinin in microplate wells coated with a cytokinin-ovalbumin conjugate. The method measured competition between free cytokinin and the bound conjugate for reaction with monoclonal anticytokinin antibodies and used a standard curve prepared by use of known amounts of free cytokinin to quantify hormone levels in unknown samples. Standard curves which consisted of logit/log plots of fluorescence units versus picomoles of competing cytokinin measured from 0.03 to 256 pmol (approximately 10-85,000 picograms) of zeatin riboside (ZR) or isopentenyl adenosine. The fluorescence ELISA was compared with radioimmunoassay for the quantification of ZR in wheat (Triticum aestivum L., cultivar Stephens) seed samples. This fluorescence ELISA method is recommended for use in combination with a fractionation method, such as HPLC, to quantify cytokinins present in plant extracts.