PRODUCTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES AGAINST ASPARTATE AMINOTRANSFERASE-P1 FROM LUPIN ROOT-NODULES

Six hybridoma clones were obtained that secreted monoclonal antibodies against the aspartate aminotransferase-Pr (AAT-P-1) isoenzyme from ro ot nodules of Lupinus angustifolius [L.] cv Uniharvest. This enzyme is found constitutively in the plant cytosol fraction. The monoclonal an tibodies produced were all of the immunoglobulin G(1) class, recognize d two distinct epitopes on the protein, and represented the major para topes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P-1 protein. One of these epitope s was unique to lupin nodule AAT-P-1. The other epitope was shown to b e present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers. Both epitopes were recogni zed by the appropriate monoclonal antibodies in both their native and denatured forms. None of the monoclonal antibodies produced reacted wi th Rhizobium lupini NZP2257, Escherichia coli extracts, or with the in ducible aspartate aminotransferase-P-2 (AAT-P-2) isoform also found in root nodules. A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was de veloped and was capable of quantitating AAT-P-1 in plant extracts. The limit of detection of AAT-P-1 was less than 15 pg/mL and AAT-P-1 prot ein could be quantified in the range 80 to 1000 pg/mL. Using this assa y, AAT-p(1) protein was shown to remain relatively constant during nod ule development. Use of an AAT-P-2-specific monoclonal antibody that i nhibits the enzyme activity of this isoform enabled the direct determi nation of AAT-PI enzyme activity in nodule extracts. Using these assay s, specific activities of the individual isoforms were calculated; tha t of the AAT-P-1 isoform was shown to be 7.5-fold higher than that of the AAT-P-2 isoform.