Rapid preparation of peroxidase: anti-peroxidase complexes for immunocytochemical use

Soluble immune complexes of horseradish peroxidase and antibody to peroxidase (PAP) have been widely used in the 'unlabelled antibody' method for the immunocytochemical detection of cellular antigens. This paper describes a simple and rapid method for preparation of these complexes by column chromatography of a mixture of the enzyme and the IgG fraction of antiperoxidase antiserum on Sephacryl S-200. The material eluting in the void column consists of stable soluble PAP complexes, with a molar peroxidase: antiperoxidase ratio of 0.8 and a molecular weight of approximately 400,000. When tested immunocytochemically this material gives identical results to those obtained with conventionally prepared PAP.