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.
Yoshimura
K
K
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
Ishikawa
T
T
Wada
K
K
Takeda
T
T
Kamata
Y
Y
Tada
T
T
Nishimura
K
K
Nakano
Y
Y
Shigeoka
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D060387:Ascorbate Peroxidases; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics; D002846:Chromatography, Affinity; D003429:Cross Reactions; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010544:Peroxidases / Q000276:immunology* / Q000302:isolation & purification; D010940:Plant Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D018724:Spinacia oleracea
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; D060387:Ascorbate Peroxidases