A simple method for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli.

A simple method was described for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli. IgG was subjected to successive processes of pepsin digestion, reduction with 2-mercaptoethylamine, affinity-purification and reaction with maleimide groups introduced into the enzymes. In the present method, gel filtration was required only once to separate the conjugate from unconjugated components in the final step, while gel filtration had to be repeated 3-4 times in the previous methods. The conjugate preparations obtained by the present method contained less nonspecific conjugate and gave a lower background by immunoenzymometric assay technique than those obtained by the previous method.