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 epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region 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 inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.