Production and characterisation of monoclonal antibodies to a cationic peroxidase from spinach leaves

Abstract

Five different murine monoclonal antibodies specific for a cationic isoperoxidase (C1) prepared from spinach leaves have been produced and characterised. Immunoassays showed that they have different affinities for the purified C1 and that they recognise different epitopes of the peroxidase molecule. Immunoaffinity chromatography, using McAbs-Sepharose columns, has shown that some of the eight isoperoxidases present in a leaf extract share at least one similar epitope, recognised by one McAb. An immunoassay was developed that permits quantitation of purified C1. Two McAbs seem strictly specific for C1, but none of the five McAbs binds proteins other than peroxidases, suggesting that they could all be directed against the peptidic part of the glycoprotein. One McAb specific for C1 also strongly inhibits its enzyme activity, but has no effect on the activity of the other isoperoxidases. Kinetic studies have shown that the inhibition occurs very rapidly after the addition of the McAb, either in classical peroxidase reaction, or during the peroxidase-mediated oxidation of NADH.