A comparative study of horseradish peroxidase conjugates prepared with a one step and a two step method

Abstract

The authors compared horse radish peroxidase (HRP) labeled rabbit antihuman immunoglobulin G (IgG) conjugates prepared by a onestep and a 2 step method. Glutaraldehyde was used as a cross linking agent. Two methods were used for removing unconjugated HRP: sephadex G200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a 2 step method. Removing unconjugated HRP improved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by sephadex G200 gel chromatography.