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-P1 (AAT-P1) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. This enzyme is found constitutively in the plant cytosol fraction. The monoclonal antibodies produced were all of the immunoglobulin G1 class, recognized two distinct epitopes on the protein, and represented the major paratopes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P1 protein. One of these epitopes was unique to lupin nodule AAT-P1. The other epitope was shown to be present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers. Both epitopes were recognized by the appropriate monoclonal antibodies in both their native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257, Escherichia coli extracts, or with the inducible aspartate aminotransferase-P2 (AAT-P2) isoform also found in root nodules. A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was developed and was capable of quantitating AAT-P1 in plant extracts. The limit of detection of AAT-P1 was less than 15 pg/mL and AAT-P1 protein could be quantified in the range 80 to 1000 pg/mL. Using this assay, AAT-P1 protein was shown to remain relatively constant during nodule development. Use of an AAT-P2-specific monoclonal antibody that inhibits the enzyme activity of this isoform enabled the direct determination of AAT-P1 enzyme activity in nodule extracts. Using these assays, specific activities of the individual isoforms were calculated; that of the AAT-P1 isoform was shown to be 7.5-fold higher than that of the AAT-P2 isoform.
Jones
W. T.
WT
Plant Improvement Division, The Horticulture and Food Research Institute of New Zealand, Private Bag 11-030, Palmerston North, New Zealand.
Jones
S. D.
SD
Harvey
D.
D
Rodber
K. R.
KR
Ryan
G. B.
GB
Reynolds
PHS.
P