Measurement of Aspartate Aminotransferase-P₂ in Developing Lupin Nodules Using a Two Site ELISA Employing Monoclonal Antibodies

Abstract

Leguminous root nodules contain two forms of aspartate aminotransferase (AAT). AAT-P1 is con- stitutively expressed in roots and nodules, whereas the second isoenzyme, AAT-P₂, is induced in developing nodules. A two site ELISA system was developed to quantify AAT-P₂ in nodule extracts. The assay comprised two affinity-purified monoclonal antibodies directed against different epitopes on the AAT-P₂ molecule. The sensitivity was less than 200 pg mL^-1, and AAT-P₂ could be quantified in the range 1 to 15 ng mL^-1. The assay was specific for AAT-P₂, and did not measure AAT-P₁. The assay was used to specifically measure AAT-P₂ in the developing legume root nodule formed by the symbiotic liaison between Rhizobium lupini NZP2257 and the roots of Lupinus angustifolius L. The ELISA method, which measures AAT-P2 protein, was contrasted with an approach which relied on the heat lability of AAT-P₂ at 60°C to measure individual isoenzyme catalytic activity.