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 prodrug-based immunotherapeutic applications. These antibodies may be polyclonal, monoclonal, bispecific or genetically engineered in origin. Often, purification of the antibody is the single greatest obstacle to obtaining immunoprobes with high specific activity [Milstein and Cuello, Nature 305 (1983) 537]. We have circumvented this problem by utilising benzhydroxamic acid-agarose to purify the antibodies tagged with HRPO as a preformed immune complex. Benzhydroxamic acid has been shown to have affinity for the active site of HRPO [de Ropp et al., Biochemistry 38 (1999) 1077]. A preliminary ammonium sulfate precipitation of 250 ml 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 affinity column which was washed, and the labelled bispecfic monoclonal antibodies were eluted under mild conditions (borate buffer pH 9.0). The effective yield of this bispecific antibody-HRPO complex was 30 assay plates or 3000 wells. We have also successfully co-purified covalent polyclonal-HRPO conjugates and HRPO-labelled streptavidin using a similar strategy to obtain enzyme-labelled probes with high specific activities for a multitude of applications.