Preparation of a Monomeric 2, 4-Dinitrophenyl Fab'-β-D-Galactosidase Conjugate for Immunoenzymometric Assay

Abstract

A method is described for the preparation of a monomeric Fab'-β-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')₂ was labeled with 2, 4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into β-D-galactosidase through thiol groups using N, N'-o-phenyl-enedimaleimide. The monomeric 2, 4-dinitrophenyl Fab'-β-D-galactosidase conjugate was subsequently separated from unconjugated β-D-galactosidase by affinity chromatography on a column of (anti-2, 4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of β-D-galactosidase activity was asso-ciated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.