Development of an ELISA for estimation of the copper nutritional status of wheat and cotton

Studies were carried out with hydroponically grown wheat and cotton to develop the Cu-requiring protein phenolase as a biomarker of Cu nutrient status. Isozymes of phenolase whose levels were reduced by Cu deficiency were identified by Western blots. A competitive enzyme-linked, immunosorbent assay (ELISA) was developed that could detect as little as 25 ng of phenolase. The ELISA revealed that Cu-sufficient cotton leaves had about 4-fold more phenolase antigen than did Cu-sufficient wheat leaves. In both species, the level of phenolase was reduced by 2- to 5-fold in leaves of Cu-deficient plants. Because the immunoassay for phenolase protein is rapid, inexpensive, and can be carried out with small amounts of leaf material, it has potential as a tool for assessment of the Cu status of crop plants.Abbreviations ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - TBS Trisbuffered saline (20 mM Tris-HCl, pH 9.5, 150 mM NaCl) - TBST Tris-buffered saline containing 0.05% Tween-20