A general affinity method to purify peroxidase-tagged antibodies

Antibodies tagged with enzymes, e,g. horseradish peroxidase (HRPO) are used extensively in a broad range of immunoassay, immunohistochemical, and prod rug-based immunotherapeutic applications. These antibodies may be polyclona l, monoclonal, bispecific or genetically engineered in origin. Often, purif ication of the antibody is the single greatest obstacle to obtaining immuno probes with high specific activity [Milstein and Cuello, Nature 305 (1983) 537]. We have circumvented this problem by utilising benzhydroxamic acid-ag arose to purify the antibodies tagged with HRPO as a preformed immune compl ex. Benzhydroxamic acid has been shown to have affinity for the active site of HRPO [de Ropp et al., Biochemistry 38 (1999) 1077]. A preliminary ammon ium sulfate precipitation of 250 mi of bispecific antibody supernatant was performed and the pellet resuspended and dialysed against phosphate buffer (pH 7.0). This fraction was incubated with HRPO, then loaded on the affinit y column which was washed, and the labelled bispecfic monoclonal antibodies were eluted under mild conditions (borate buffer pH 9.0). The effective yi eld of this bispecific antibody-HRPO complex was 30 assay plates or 3000 we lls. We have also successfully co-purified covalent polyclonal-HRPO conjuga tes and HRPO-labelled streptavidin using a similar strategy to obtain enzym e-labelled probes with high specific activities for a multitude of applicat ions. (C) 2001 Elsevier Science B.V. All rights reserved.