Characterization of monoclonal antibodies against ascorbate peroxidase isoenzymes: purification and epitope-mapping using immunoaffinity column chromatography

We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX ) isoenzymes to analyze the cross-reactivity and the structure of the epito pes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but non cross-reacted with the others. I mmunoreactive fragments in proteolytic recombinant APX isoenzymes were dete cted by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His reg ion obtained by the lysyl endopeptidase digestion. The chl-mAb6 was capable of binding to the fragment. D-I-K-E-K-R, which is consistent with an inher ent region of chloroplastic isoenzymes. No fragments reacting to the chl-mA b3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, whi ch may be destroyed by the enzymatic cleavage. We concluded that the peptid es identified as epitope are characteristic evidence of monoclonal antibodi es. (C) 2001 Published by Elsevier Science B.V.