Characterization of Antigenic Properties of Horseradish Peroxidase with Monoclonal Antibodies

O. V. Ignatenko^*, M. Yu. Rubtsova, T. V. Cherednikova, I. V. Ouporov, and A. M. Egorov

Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 939-2742; E-mail: ovig@enz.chem.msu.ru

^* To whom correspondence should be addressed.

Received January 14, 2002; Revision received February 7, 2002

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Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO₁ and 36F₉ were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.

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KEY WORDS: antigenic determinants, horseradish peroxidase, epitope mapping, PEPSCAN, monoclonal antibodies

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