Conjugation of Glucose Oxidase from Aspergillus niger and Rabbit Antibodies Using N-Hydroxysuccinimide Ester of N-(4-Carboxycyclohexylmethyl)-Maleimide

Abstract

Glucose oxidase from Aspergillus niger was conjugated with rabbit immunoglobulin G or its monovalent fragments (Fab'). The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide to introduce maleimide groups, which were then allowed to react with thiol groups of reduced IgG or Fab'. More than 40% of immunoglobulin G, Fab' and enzyme used could be conjugated without self-coupling. The enzyme activity decreased about 26 and 15% upon conjugation with immunoglobulin G and Fab', respectively, and the ability of antibody to bind to antigen was well preserved in conjugates. Conjugate preparations purified by gel filtration contained little free form of immunoglobulin G, Fab' or enzyme. Both the cross-link and enzyme activity in Fab' conjugate were stable at pH 6-7 at 4 degrees C for at least 6 months.