Preparation of Monomeric Affinity-Purified Fab'-ß-D-Galactosidase Conjugate for Immunoenzymometric Assay

A Simpler method for the preparation of monomeric affinity-purified Fab'-ß-D-galactosidase conjugate is described. Rabbit (anti-human IgG) serum was subjected to successive processes of pepsin digestion to convert IgG to F(ab')2′ reduction with 2-mercaptoethy on a column of human IgG-Sepharose 4B. The affinity-purified Fab' thus obtained without using gel filtration was reacted with excess of maleimide groups introduced into ß-D-galactosidase from Escherichia coli. The monomeric Fab'-ß-D-galactosidase conjugate formed was separated from unconjugated Fab' by gel filtration and from unconjugated ß-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B. By immunoenzymometric assay technique for human IgG, the monomeric conjugate was compared with a monomeric conjegate prepared by a previously reported complexmethod and non-monomeric conjugate which contained 3.7 Fab' molecules per ß-D-galactosidase molecule. The present monomeric conjugate provided as sensitive a dose-response curve as the previously reported monomeric conjugate and a more sensitive dose-response curve than the non-monomeric conjugate.