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

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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 associated 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.