A new covalent peroxidase conjugation method using bis(sulfosuccinimidyl) suberate as cross-linking reagent in a two-step procedure

ABSTRACT A new method has been developed to prepare horseradish peroxidase (HRP) conjugates using bis(sulfosuccinimidyl)suberate (BS3) as cross-linking reagent in a two-step procedure. The enzyme is reacted with BS3, introducing active ester molecules into the enzyme itself, and it is then used directly to label amino-containing compounds without further treatment. The proteins involved in the conjugation undergo minimal modifications. The reaction conditions are evaluated, as well as studies on the conservation of the biological activities of the conjugated proteins and the stability of the conjugates in time. These conjugates are found to be significantly improved compared with similar products prepared by conventional methods.